Prostate Cancer Prognostic Compositions and Kits

Abstract

Described herein are method, compositions and kits for prognosis of prostate cancer. The methods include determining the ratio of PCA3 and of a prostate-specific marker expression in a urine sample and correlating the value of the PCA3/prostate-specific marker ratio with the aggressiveness and mortality risk of prostate cancer in the subject. The method for prognosing prostate cancer in a sample of a patient includes assessing the amount of a prostate cancer specific PCA3 mRNA and the amount of prostate-specific marker in the sample; determining a ratio value of this amount of prostate cancer specific PCA3 mRNA over the amount of prostate-specific marker; comparing the ratio value to at least one predetermined cut-off value, wherein a ratio value above the predetermined cut-off value is indicative of a higher risk of mortality of prostate cancer as compared to a ratio value below the predetermined cut-off value.

1 .- 26 . (canceled) 
     
     
         27 . A method of characterizing the relative amount of PCA3 nucleic acid in a urine sample obtained from a human subject, the method comprising the steps of:
 (a) extracting nucleic acids from the urine sample;   (b) quantifying, with a real-time nucleic acid amplification reaction that uses nucleic acids extracted in step (a) or the complements thereof as templates, amounts of   (i) a PCA3 nucleic acid, and   (ii) a second marker nucleic acid; and   (c) determining a ratio value for the quantified amount of the PCA3 nucleic acid over the quantified amount of the second marker nucleic acid, thereby characterizing the relative amount of the PCA3 nucleic acid in the urine sample,   wherein the real-time nucleic acid amplification reaction uses a pair of primers and a DNA polymerase to amplify the PCA3 nucleic acid, and   wherein the real-time nucleic acid amplification reaction comprises monitoring synthesis of the PCA3 nucleic acid with an oligonucleotide probe having a detectable label covalently attached thereto.   
     
     
         28 . The method of  claim 27 ,
 wherein step (a) comprises extracting RNA from the urine sample, and   wherein prior to conducting step (b) there is a step for reverse transcribing extracted RNA from step (a) to synthesize cDNA templates used in the real-time nucleic acid amplification reaction.   
     
     
         29 . The method of  claim 27 ,
 wherein a primer of the pair of primers comprises a base sequence that hybridizes to an exon-exon junction in spliced PCA3 RNA or the complement thereof.   
     
     
         30 . The method of  claim 29 , wherein a first primer of the pair of primers comprises a base sequence complementary to an exon sequence present in PCA3 RNA, the exon sequence being selected from the group consisting of: an exon 4a sequence, an exon 4b sequence, an exon 4c sequence, and an exon 4d sequence. 
     
     
         31 . The method of  claim 30 ,
 wherein a second primer of the pair of primers hybridizes to a polymerase-dependent extension product of the first primer of the pair of primers using nucleic acids extracted in step (a) as templates,   wherein the polymerase-dependent extension product comprises a base sequence complementary to a base sequence contained within PCA3 exon 3, and   wherein the second primer hybridizes to the base sequence complementary to the base sequence contained within PCA3 exon 3.   
     
     
         32 . The method of  claim 31 , wherein either the first primer comprises a sequence of 3′-terminal bases that hybridize to a PCA3 exon 3 sequence, or the second primer comprises a sequence of 3′-terminal bases that hybridize to the complement of a PCA3 exon 4a sequence. 
     
     
         33 . The method of  claim 32 , wherein the second marker nucleic acid is selected from the group consisting of HK2/KLK2 nucleic acid, PSMA nucleic acid, transglutaminase 4 nucleic acid, acid phosphatase nucleic acid, PCGEM1 nucleic acid, NKX3.1 nucleic acid, prostate stem cell antigen (PSCA) nucleic acid, prostate tumor inducing gene-1 (PTI-1) nucleic acid, PDEF nucleic acid, TMPRSS2 nucleic acid, and ProStase nucleic acid. 
     
     
         34 . The method of  claim 29 ,
 wherein the exon-exon junction in spliced PCA3 RNA or the complement thereof is a junction joining PCA3 exon 3 to PCA3 exon 4 in spliced PCA3 RNA,   wherein the first primer hybridizes at its 5′-end to a sequence of bases contained within PCA3 exon 4, and   wherein the first primer hybridizes at its 3′-end to a sequence of bases contained within PCA3 exon 3.   
     
     
         35 . The method of  claim 34 ,
 wherein the second primer hybridizes to a polymerase-dependent extension product of the first primer using nucleic acids extracted in step (a) as templates,   wherein the polymerase-dependent extension product comprises a base sequence complementary to a base sequence contained within PCA3 exon 3, and   wherein the second primer hybridizes to the base sequence complementary to the base sequence contained within PCA3 exon 3.   
     
     
         36 . The method of  claim 27 , wherein the real-time nucleic acid amplification reaction comprises temperature cycling steps. 
     
     
         37 . The method of  claim 27 , wherein the detectable label of the oligonucleotide probe is a fluorescent moiety, and wherein the oligonucleotide probe further comprises a quencher moiety covalently attached thereto. 
     
     
         38 . The method of  claim 27 ,
 wherein the real-time nucleic acid amplification reaction produces a double-stranded amplification product,   wherein a first strand of the double-stranded amplification product comprises each of a base sequence present in PCA3 exon 3 and a base sequence present in PCA3 exon 4, and   wherein a second strand of the double-stranded amplification product comprises each of the complement of the base sequence present in PCA3 exon 3 and the complement of the base sequence present in PCA3 exon 4.   
     
     
         39 . The method of  claim 38 , wherein one primer of the pair of primers hybridizes to the complement of the base sequence present in PCA3 exon 3. 
     
     
         40 . The method of  claim 38 , wherein a first primer of the pair of primers hybridizes to a base sequence present in PCA3 exon 4a. 
     
     
         41 . The method of  claim 40 , wherein a second primer of the pair of primers hybridizes to the complement of the base sequence present in PCA3 exon 3. 
     
     
         42 . The method of  claim 27 , wherein the PCA3 nucleic acid quantified in step (b) comprises a base sequence present in PCA3 exon 3 and a base sequence present in PCA3 exon 4a. 
     
     
         43 . The method of  claim 42 ,
 wherein a first primer of the pair of primers hybridizes to a PCA3 exon 4a sequence of a template nucleic acid extracted in step (a) and extends by activity of the DNA polymerase to produce a polymerase-dependent extension product, and   wherein a second primer of the pair of primers hybridizes to the complement of a PCA3 exon 3 sequence contained in the polymerase-dependent extension product.   
     
     
         44 . The method of  claim 43 , wherein the second marker nucleic acid is selected from the group consisting of HK2/KLK2 nucleic acid, PSMA nucleic acid, transglutaminase 4 nucleic acid, acid phosphatase nucleic acid, PCGEM1 nucleic acid, NKX3.1 nucleic acid, prostate stem cell antigen (PSCA) nucleic acid, prostate tumor inducing gene-1 (PTI-1) nucleic acid, PDEF nucleic acid, TMPRSS2 nucleic acid, and ProStase nucleic acid. 
     
     
         45 . The method of  claim 28 ,
 wherein each primer of the pair of primers hybridizes to a sequence contained within a different PCA3 exon or the complement thereof,   wherein a first primer of the pair of primers hybridizes to a template nucleic acid extracted in step (a) or the complement thereof and extends by activity of the DNA polymerase to produce a polymerase-dependent extension product, and   wherein a second primer of the pair of primers hybridizes to the polymerase-dependent extension product of the first primer.   
     
     
         46 . The method of  claim 45 , wherein the first primer comprises a base sequence complementary to an exon sequence present in PCA3 RNA, the exon sequence being selected from the group consisting of: an exon 4a sequence, an exon 4b sequence, an exon 4c sequence, and an exon 4d sequence. 
     
     
         47 . The method of  claim 46 , wherein the second marker nucleic acid is selected from the group consisting of HK2/KLK2 nucleic acid, PSMA nucleic acid, transglutaminase 4 nucleic acid, acid phosphatase nucleic acid, PCGEM1 nucleic acid, NKX3.1 nucleic acid, prostate stem cell antigen (PSCA) nucleic acid, prostate tumor inducing gene-1 (PTI-1) nucleic acid, PDEF nucleic acid, TMPRSS2 nucleic acid, and ProStase nucleic acid. 
     
     
         48 . The method of  claim 27 , wherein the human subject is known to have prostate cancer prior to conducting step (a). 
     
     
         49 . The method of  claim 27 , wherein if the human subject has prostate cancer, then the ratio value determined in step (c) is indicative of the aggressiveness of the prostate cancer. 
     
     
         50 . The method of  claim 27 , wherein the urine sample is a urine sample obtained without a digital rectal examination. 
     
     
         51 . The method of  claim 27 , wherein the second marker nucleic acid is selected from the group consisting of HK2/KLK2 nucleic acid, PSMA nucleic acid, transglutaminase 4 nucleic acid, acid phosphatase nucleic acid, PCGEM1 nucleic acid, NKX3.1 nucleic acid, prostate stem cell antigen (PSCA) nucleic acid, prostate tumor inducing gene-1 (PTI-1) nucleic acid, PDEF nucleic acid, TMPRSS2 nucleic acid, and ProStase nucleic acid. 
     
     
         52 . The method of  claim 27 , wherein the oligonucleotide probe used for monitoring synthesis of the PCA3 nucleic acid comprises a sequence of at least 10 consecutive nucleotides of SEQ ID NO:1, or the complement thereof. 
     
     
         53 . The method of  claim 27 ,
 wherein the real-time nucleic acid amplification reaction uses a pair of primers and the DNA polymerase to amplify the second marker nucleic acid,   wherein the real-time nucleic acid amplification reaction comprises monitoring synthesis of the second marker nucleic acid with an oligonucleotide probe having a detectable label covalently attached thereto, and   wherein the detectable label attached to the oligonucleotide probe used for monitoring synthesis of the second marker nucleic acid is different from the detectable label attached to the oligonucleotide probe used for monitoring synthesis of the PCA3 nucleic acid.