WO2009140741A1

WO2009140741A1 - Agents and methods for diagnosing the presence or risk of prostate cancer

Info

Publication number: WO2009140741A1
Application number: PCT/AU2009/000651
Authority: WO
Inventors: Martin Lavin
Original assignee: The University Of Queensland
Priority date: 2008-05-23
Filing date: 2009-05-25
Publication date: 2009-11-26

Abstract

The present invention discloses methods and agents for diagnosing the presence or risk development of prostate cancer in a subject. More particularly, the present invention discloses the use of certain PCA3 polynucleotides as markers for prostate cancer including metastatic prostate cancer. The invention has practical use in early diagnosis of prostate cancer, and in enabling better treatment and management decisions to be made in clinically and sub-clinically affected subjects.

Description

AGENTS AND METHODS FOR DIAGNOSING THE PRESENCE OR RISK OF PROSTATE CANCER

FIELD OF THE INVENTION

[0001] This invention relates generally to methods and agents for diagnosing the presence or risk development of prostate cancer including metastatic prostate cancer in subjects. The invention has practical use in early diagnosis of prostate cancer, and in enabling better treatment and management decisions to be made in clinically and sub-clinically affected subjects.

BACKGROUND OF THE INVENTION [0002] There are a number of features of prostate cancer that make early detection, monitoring, early intervention and informed management of affected mammals clinically and economically important. For example, prostate cancer is the second most common malignant cause of death worldwide in males after lung cancer and at the age of 50, a man has a greater than 40% chance of developing prostate cancer and nearly a 3% chance of dying from the disease.

[0003] Most prostate cancers initially occur in the peripheral zone of the prostate gland, away from the urethra. Tumours within this zone may not produce any symptoms, and as a result, most men with early-stage prostate cancer will not present clinical symptoms of the disease until significant progression has occurred. Overwhelming clinical evidence shows that human prostate cancer has the propensity to metastasise to bone, and the disease appears to progress inevitably from androgen dependent to castrate resistant status, leading to an increased risk of patient mortality. With very few exceptions, metastatic disease from carcinoma is fatal.

[0004] Until relatively recently, serum prostate specific antigen (PSA) measurements were regarded as the best conventional serum marker available to detect prostate cancer. However, it was discovered that despite its adequate sensitivity, the use of PSA is limited by a significant lack of specificity (Pannek, J and Partin, A. W., 1997, Oncology, 11: 1273-1278). Consequently, the clinical assessment of patients with an elevated PSA value results in the performance of unnecessary prostatic biopsies in a substantial number of men, leading to the need for better prostate cancer markers. [0005] Microarray technology has accelerated the study of the molecular events involved in prostate cancer, offering the prospect of more precise prognosis and new therapeutic strategies. The expression and function of numerous genes have been shown to be altered in prostate cancer. For example, one such gene is prostate cancer antigen 3 (PCA3), previously known as DD3 (Bussemakers, PCT/CA98/00346, published as WO 98/45420; Schalken, 1998, Eur Urol, 34 (suppl. 3): 3-6; Bussemakers et al, 1999, Cancer Research, 59: 5975-5979 and Bussemakers, 1999, Eur Urol, 35: 408-412). Higher levels of PCA3 have been observed in cancer cells as opposed to normal prostate cells (de Kok, et al. , 2002, Cancer Research, 62(9): 2695-2698). PCA3 is highly over-expressed in prostate cancer tissue in comparison to adj acent non-malignant prostatic tissue.

[0006] PC A3 is located on chromosome 9 and more precisely to region 9q21-22. It is well established in the art that PC A3 consists of four exons, which give rise, by both alternative splicing (of exon 2) and alternative polyadenylation (at three different positions in exon 4), to differently sized transcripts. However, in vitro transcription and translation of the full-length PC A3 cDNA have not provided evidence that a protein is produced and as such, the gene is thought to function as a non-coding RNA (Kok et al, 2002, Can Res, 62: 2695- 2698). hi addition to the transcripts, the promoter of the PC A3 gene has been thoroughly studied (US 6, 897,024).

[0007] U.S. Patent No. 7,008,765 discloses a method of detecting PCA3 nucleic acids in a sample to diagnose, assess or prognose a mammal afflicted with prostate cancer. This patent discloses in Example 2 that semi-quantitative RT-PCR analysis of PCA3 expression allowed a clear distinction between benign and malignant specimens in 23 of 25 cases, however, no results are shown. Although this assay has high specificity, the semiquantitative nature of the assay makes it cumbersome. Van Gils et al., 2007 {Clinical Cancer Research, 13(3): 939-943) performed a multi-centre study to evaluate the diagnostic performance of the PC A3 urine test for detecting prostate cancer. The sensitivity of the PC A3 test was determined to be 65% and the specificity was 66% (versus 47% for serum PSA). However, the replicability of the PC A3 test has yet to be established. This prior art assay suffers from several drawbacks, including moderate specificity and sensitivity, as well as relying on quantitative PCR analysis, which is both costly and time-consuming.

[0008] The present invention is predicated in part on the discovery that specific PCA3 splice variants are more useful than others in differentiating between prostate cancer and/or metastatic prostate cancer and benign prostatic hyperplasia (BPH) or normal prostate tissue. In particular, the present inventors have discovered that in addition to the four exons known in the art (see Figure 1), the PC A3 gene comprises two additional exons (referred to herein as "exon 2a" as set forth in SEQ ID NO: 6 and "exon 2b" as set forth in SEQ ID NO: 7) which are located between archetypal exons 1 and 2 (see Figure 2). Transcripts containing novel exons 2a and/or 2b and/or archetypal exon 2 (referred to herein as "exon 2c") are differentially expressed between prostate cancer patients or patients with prostate cancer metastases and normal prostatic subjects or BPH prostatic subjects. The inventors have determined unexpectedly that some embodiments of these assays, which merely rely on detecting the presence of such transcripts, provide high specificities ( « 85%) of diagnosing prostate cancer. These assays are thus advantageous over the prior art PC A3 assays which rely on laborious quantitative or semi-quantitative techniques. The inventors have reduced these discoveries to practice in novel diagnostic assays, as described hereafter.

[0009] Additionally, the present inventors have discovered four novel transcription start sites upstream of the archetypal transcription start site of the PC A3 gene, which is located at nucleotide 1151 of the sequence set forth in SEQ ID NO: 15. The novel transcription start sites are located at nucleotides 1 (also referred to herein as PCA3-3), 452 (also referred to herein as PC A3 -4), 511 (also referred to herein as PCA3-5), and 1015 (also referred to herein as PCA3-6), of the sequence set forth in SEQ ID NO: 15 and extend the size of the prototypic PCA3 exon 1 (also referred to herein as exon Ia) by 1150, 699, 640 and 136 nucleotides, respectively. Accordingly, the novel transcription start sites define four novel exon 1 variants as follows: (1) a 256-nucleotide exon 1 variant (also referred to herein as exon Ib), which includes nucleotides 1015 to 1270 of the sequence set forth in SEQ ED NO: 15; (2) a 760-nucleotide exon 1 variant (also referred to herein as exon Ic), which includes nucleotides 511 to 1270 of the sequence set forth in SEQ ID NO: 15; (3) an 819-nucleotide exon 1 variant (also referred to herein as exon Id), which includes nucleotides 452 to 1270 of the sequence set forth in SEQ ID NO: 15; and (4) a 1270-nucleotide exon 1 variant (also referred to herein as exon Ie), which includes nucleotides 1 to 1270 of the sequence set forth in SEQ ID NO: 15. In accordance with the present invention, the novel sequences upstream of the archetypal transcription start site can be used to design probes or primers for detecting the diagnostic markers of the present invention. SUMMARY OF THE INVENTION

[0010] The present invention represents a significant advance over current technologies by providing better surrogate markers of prostate cancer including metastatic prostate cancer as well as facile methods of diagnosing or detecting the risk of developing prostate cancer or prostate cancer metastasis. The present invention discloses methods of diagnosing the presence or risk of development of prostate cancer and prostate cancer metastasis in a subject through the detection in biological tissue samples of exon la- containing PCA3 transcripts, exon 2b-containing PCA3 transcripts and/or exon 2c-containing PC A3 transcripts. The methods of the invention can be used for diagnosing prostate cancer including metastatic prostate cancer in subjects with or without symptoms of this disease.

[0011] Accordingly, in one aspect, the present invention provides methods for diagnosing the presence or risk of development of prostate cancer or prostate cancer metastasis in a subject, wherein the methods generally comprise detecting in the subject expression of a PC A3 nucleic acid sequence comprising an exon 2a sequence, as set forth for example in SEQ ID NO: 6, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 6, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 6 under at least high stringency conditions. In accordance with the present invention, expression in the subject of a PC A3 nucleic acid sequence comprising an exon 2a sequence, is indicative of the presence or risk of development of prostate cancer and/or metastasis or a negative prognosis. By contrast, expression in the subject of a PCA3 nucleic acid sequence lacking an exon 2a sequence is indicative of the absence of prostate cancer and/or metastasis or a positive prognosis. In some embodiments, expression in the subject of a PC A3 nucleic acid sequence lacking an exon 2a sequence and an exon 2b sequence is indicative of the absence of prostate cancer and/or metastasis or a positive prognosis. In some embodiments, expression in the subject of a PC A3 nucleic acid sequence lacking an exon 2a sequence, an exon 2b sequence and an exon 2c sequence is indicative of the absence of prostate cancer and/or metastasis or a positive prognosis, hi some embodiments, expression in the subject of a PC A3 nucleic acid sequence lacking an exon 2a sequence and an exon 2c sequence is indicative of the absence of prostate cancer and/or metastasis or a positive prognosis.

[0012] The PCA3 nucleic acid sequence that is detected according to this aspect will typically contain one or more additional exons. For example, the expressed nucleic acid sequence may further comprise an exon 1 sequence as for example set forth in SEQ ID NO: 1, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 1, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 1 under at least high stringency conditions; (2) an exon Ib sequence, as for example set forth in SEQ ID NO: 2, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 2, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 2 under at least high stringency conditions; (3) an exon Ic sequence, as for example set forth in SEQ ID NO: 3, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 3, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 3 under at least high stringency conditions; (4) an exon Id sequence, as for example set forth in SEQ ED NO: 4, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 4, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 4 under at least high stringency conditions; and (5) an exon Ie sequence, as for example set forth in SEQ ID NO: 5, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 5, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 5 under at least high stringency conditions. In some embodiments, the expressed nucleic acid sequence further comprises an exon 2b sequence, as set forth for example in SEQ ID NO: 7. hi some embodiments, the expressed nucleic acid sequence further comprises an exon 2c sequence, as set forth for example in SEQ ID NO: 8. In some embodiments, the expressed nucleic acid sequence further comprises an exon 3 sequence, as set forth for example in SEQ ID NO: 9. In some embodiments, the expressed nucleic acid sequence further comprises an exon 4 sequence, as set forth for example in SEQ ID NO: 10. In specific embodiments, the expressed nucleic acid sequence further comprises an exon 1 sequence, an exon 2b sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2a-exon 2b-exon 2c-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 11-15. hi other embodiments, the expressed nucleic acid sequence further comprises an exon 1 sequence, an exon 2b sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2a-exon 2b-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 16-20. As used herein, reference to "an exon 1 sequence" includes and encompasses a sequence selected from exon Ia, exon Ib, exon Ic, exon Id and exon Ie. hi other embodiments, the expressed nucleic acid sequence further comprises an exon 1 sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exoh 2a-exon 2c-exon 3 -exon 4), illustrative examples of which are set forth in SEQ ID NO: 21-25. In other specific embodiments, the expressed nucleic acid sequence further comprises an exon 1 sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2a-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 31-35.

[0013] In another aspect, the present invention provides methods for diagnosing the presence or risk of development of prostate cancer or prostate cancer metastasis in a subject, wherein the methods generally comprise detecting in the subject expression of a PC A3 nucleic acid sequence comprising an exon 2b sequence, as set forth for example in SEQ ID

NO: 7, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 7, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 7 under at least high stringency conditions, hi accordance with the present invention, expression in the subject of a PCA3 nucleic acid sequence comprising an exon 2b sequence, is indicative of the present or risk of development of prostate cancer and/or metastasis or a negative prognosis. By contrast, expression in the subject of a PC A3 nucleic acid sequence lacking an exon 2b sequence is indicative of the absence of prostate cancer and/or metastasis or a positive prognosis. In some embodiments, expression in the subject of a PC A3 nucleic acid sequence lacking an exon 2b sequence and an exon 2a sequence is indicative of the absence of prostate cancer and/or metastasis or a positive prognosis. In some embodiments, expression in the subject of a PCA3 nucleic acid sequence lacking an exon 2b sequence, an exon 2a sequence and an exon 2c sequence is indicative of the absence of prostate cancer and/or metastasis or a positive prognosis. In some embodiments, expression in the subject of a PCA3 nucleic acid sequence lacking an exon 2b sequence and an exon 2c sequence is indicative of the absence of prostate cancer and/or metastasis or a positive prognosis.

[0014] The PCA3 nucleic acid sequence that is detected according to this aspect will typically contain one or more additional exons. For example, the expressed nucleic acid sequence may further comprise an exon 1 sequence, illustrative examples of which include: (a) an exon Ia sequence, as for example set forth in SEQ ID NO: 1, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 1, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 1 under at least high stringency conditions; (2) an exon Ib sequence, as for example set forth in SEQ ID NO: 2, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 2, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 2 under at least high stringency conditions; (3) an exon Ic sequence, as for example set forth in SEQ ID NO: 3, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 3, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 3 under at least high stringency conditions; (4) an exon Id sequence, as for example set forth in SEQ ID NO: 4, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 4, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 4 under at least high stringency conditions; and (5) an exon Ie sequence, as for example set forth in SEQ ID NO: 5, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 5, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 5 under at least high stringency conditions. In some embodiments, the expressed nucleic acid sequence further comprises an exon 2a sequence, as set forth for example in SEQ ID NO: 6. In some embodiments, the expressed nucleic acid sequence further comprises an exon 2c sequence, as set forth for example in SEQ ED NO: 8. In some embodiments, the expressed nucleic acid sequence further comprises an exon 3 sequence, as set forth for example in SEQ ID NO: 9. In some embodiments, the expressed nucleic acid sequence further comprises an exon 4 sequence, as set forth for example in SEQ ID NO: 10. In specific embodiments, the expressed nucleic acid sequence further comprises an exon 1 sequence, an exon 2a sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2a-exon 2b-exon 2c-exon 3 -exon 4), illustrative examples of which are set forth in SEQ ID NO: 11-15. In other specific embodiments, the expressed nucleic acid sequence further comprises an exon 1 sequence, an exon 2a sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2a- exon 2b-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 16-20. In other specific embodiments, the expressed nucleic acid sequence further comprises an exon 1 sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2b-exon 2c-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 26-30. In other embodiments, the expressed nucleic acid sequence further comprises an exon 1 sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2b-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 36-40.

[0015] In yet another aspect, the present invention provides methods for diagnosing the presence or risk of development of prostate cancer or prostate cancer metastasis in a subject, wherein the methods generally comprise detecting in the subject expression of a

PCA3 nucleic acid sequence comprising an exon 2c sequence, as set forth for example in SEQ ID NO: 8, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 8, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 8 under at least high stringency conditions. In accordance with the present invention, expression in the subject of a PC A3 nucleic acid sequence comprising an exon 2c sequence, is indicative of the presence or risk of development of prostate cancer.and/or metastasis or a.negative prognosis. By contrast, expression in the subject of a PCA3 nucleic acid sequence lacking an exon 2c sequence is indicative of the absence of prostate cancer and/or metastasis or a positive prognosis. In some embodiments, expression in the subject of a PC A3 nucleic acid sequence lacking an exon 2c sequence and an exon 2a sequence is indicative of the absence of prostate cancer and/or metastasis or a positive prognosis. In some embodiments, expression in the subject of a PCA3 nucleic acid sequence lacking an exon 2c sequence, an exon 2a sequence and an exon 2b sequence is indicative of the absence of prostate cancer and/or metastases or a positive prognosis. In some embodiments, expression in the subject of a PCA3 nucleic acid sequence lacking an exon 2b sequence and an exon 2c sequence is indicative of the absence of prostate cancer and/or metastases or a positive prognosis.

[0016] The PCA3 nucleic acid sequence that is detected according to this aspect will typically contain one or more additional exons. For example, the expressed nucleic acid sequence may further comprise an exon 1 sequence, illustrative examples of which include: (a) an exon Ia sequence, as for example set forth in SEQ ID NO: 1, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 1, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 1 under at least high stringency conditions; (2) an exon Ib sequence, as for example set forth in SEQ ID NO: 2, or a sequence that displays at least 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 2, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 2 under at least high stringency conditions; (3) an exon Ic sequence, as for example set forth in SEQ ID NO: 3, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 3, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 3 under at least high stringency conditions; (4) an exon Id sequence, as for example set forth in SEQ ID NO: 4, or a sequence that displays at least 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 4, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 4 under at least high stringency conditions; and (5) an exon Ie sequence, as for example set forth in SEQ ID NO: 5, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 5, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 5 under at least high stringency conditions. In some embodiments, the expressed nucleic acid sequence further comprises an exon 2a sequence, as set forth for example in SEQ ID NO: 6. In some embodiments, the expressed nucleic acid sequence further comprises an exon 2b sequence, as set forth for example in SEQ ID NO: 7. In some embodiments, the expressed nucleic acid sequence further comprises an exon 3 sequence, as set forth for example in SEQ ID NO: 9. In some embodiments, the expressed nucleic acid sequence further comprises an exon 4 sequence, as set forth for example in SEQ ID NO: 10. In specific embodiments, the expressed nucleic acid sequence further comprises an exon 1 sequence, an exon 2a sequence, an exon 2b sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1 -exon 2a-exon 2b-exon 2c-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 11-15. In other specific embodiments, the expressed nucleic acid sequence further comprises an exon 1 sequence, an exon 2a sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2a- exon 2c-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 21-25. In other specific embodiments, the expressed nucleic acid sequence further comprises an exon 1 sequence, an exon 2b sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2b-exon 2c-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 26-30. In other embodiments, the expressed nucleic acid sequence further comprises an exon 1 sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2c-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 41-45.

[0017] As used herein, the expressed nucleic acid sequences of the invention are collectively referred to as "prostate cancer marker polynucleotides". [0018] In some embodiments, the methods comprise detecting a prostate cancer marker polynucleotide in a biological sample obtained from the subject.

[0019] In other embodiments, the methods comprise quantitating a prostate cancer marker polynucleotide in a biological sample obtained from the subject. In illustrative examples of this type, the methods comprise: (1) providing a biological sample from the subject; (2) measuring in the biological sample the level of a prostate cancer marker polynucleotide; and (3) comparing the measured level of the prostate cancer marker polynucleotide to the level of a corresponding prostate cancer marker polynucleotide in a reference sample obtained from one or more normal subjects or from one or more subjects lacking prostate cancer, wherein a difference in the level of the prostate cancer marker polynucleotide in the biological sample as compared to the level of the corresponding prostate cancer-marker polynucleotide-in the reference sample is indicative, of the presence or risk of developing prostate cancer and/or metastasis in the subject or a negative prognosis.

[0020] In some embodiments, the methods comprise: (1) providing a biological sample from the subject; (2) measuring in the biological sample the level of a prostate cancer marker polynucleotide; and (3) comparing the measured level of the prostate cancer marker polynucleotide to the level of a corresponding prostate cancer marker polynucleotide in a reference sample obtained from one or more subjects with BPH, wherein a difference in the level of the prostate cancer marker polynucleotide in the biological sample as compared to the level of the corresponding prostate cancer marker polynucleotide in the reference sample is indicative of the presence or risk of developing prostate cancer and/or metastases in the subject.

[0021] In the above embodiments, the difference in level of the prostate cancer marker polynucleotide typically represents an increase in the level of the prostate cancer marker polynucleotide as compared to the level of the corresponding prostate cancer marker polynucleotide, which is hereafter referred to as "aberrant expression." Thus, in illustrative examples of this type, the presence or risk of development of prostate cancer is determined by detecting an at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or even an at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% increase in the level of the prostate cancer marker polynucleotide, as compared to the level of the corresponding prostate cancer marker polynucleotide.

[0022] In some embodiments, the prostate cancer marker polynucleotide is the same as the corresponding prostate cancer marker polynucleotide. In other embodiments, the expression product is a variant (e.g., an allelic variant) of the corresponding prostate cancer marker polynucleotide.

[0023] In some embodiments, the biological sample comprises a tissue sample (e.g., prostatectomy specimens or TRUS biopsies). In illustrative examples of this type, the biological sample largely comprises (i.e., at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) prostate cells.

[0024] In other embodiments, the biological samples is a fluid sample. In illustrative examples of this type, the biological sample comprises prostatic fluid, seminal fluid, ejaculate fluid and peripheral blood. [0025] In some embodiments, the methods further comprise diagnosing the absence of prostate cancer or prostate cancer metastasis when the measured level of the prostate cancer marker polynucleotide is the same as or similar to the measured level of the corresponding prostate cancer marker polynucleotide, hi these embodiments, the measured level of the prostate cancer marker polynucleotide varies from the measured level of the corresponding prostate cancer marker polynucleotide by less than or no more than about 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.1%, which is hereafter referred to as "normal expression".

[0026] hi some embodiments, the prostate cancer marker polynucleotide is suitably a RNA or a DNA copy of the RNA whose level is measured using at least one nucleic acid probe that hybridizes under at least low, medium or high stringency conditions to the RNA or to the DNA copy, wherein the nucleic acid probe comprises at least 10 contiguous nucleotides of a prostate cancer marker polynucleotide. In some embodiments, the measured level or abundance of the RNA or its DNA copy is normalized to the level or abundance of a reference RNA or a DNA copy of the reference RNA that is present in the same sample. Suitably, in some embodiments, the level of nucleic acid probe that is bound to the RNA or to the DNA copy is measured by nucleic acid amplification (e.g., using polymerase chain reaction (PCR)). In other embodiments, the level of nucleic acid probe that is bound to the RNA or to the DNA copy is measured by nuclease protection assay. In still other embodiments the nucleic acid probe is immobilized on a solid or semi-solid support, hi illustrative examples of this type, the nucleic acid probe forms part of a spatial array of nucleic acid probes. In some embodiments, the level of nucleic acid probe that is bound to the RNA or to the DNA copy is measured by hybridization (e.g., using a nucleic acid array). [0027] In a related aspect, the present invention provides methods for providing a prognosis of prostate cancer in a subject comprising: (1) detecting expression or aberrant expression of a prostate cancer marker polynucleotide in the subject; (2) detecting or determining at least one factor selected from the group consisting of: the subject's pre- treatment PSA; the subject's post-treatment PSA; primary Gleason grade in a biopsy specimen obtained from the subject; secondary Gleason grade in a biopsy specimen obtained from the subject; Gleason sum in a biopsy specimen obtained from the subject; pre-radical primary therapy of the subject; total length of cancer in biopsy cores obtained from the subject; number of positive biopsy cores obtained from the subject; percent of tumour biopsy in a multiple core biopsy set obtained from the subject; primary Gleason grade in a pathological specimen obtained from the subject; secondary Gleason grade in a pathological specimen obtained from the subject; Gleason sum in a pathological specimen obtained from the subject; the subject's pre-operative TGF-ssl level; the subject's prostatic capsular invasion level (PCI; also known as extracapsular invasion or extracapsular extension); the subject's surgical margin status; the subject's seminal vesicle involvement; the subject's lymph node status; the subject's pre-operative IL6sR level; the sensitivity of the subject's cancer to hormone therapy; the resistance of the subject's cancer to hormone therapy; the subject's prior therapy and/or clinical stage; and (3) correlating (1) and (2) with disease outcome. In some embodiments, the factor is selected from the group consisting of primary Gleason grade; secondary Gleason grade; Gleason sum. Suitably, the subject's clinical stage is selected from T3a, T3, T2c, T2b, T2a, T2, Tie, TIb, TIa or Tl. In some embodiments, the subject's prior therapy is a primary therapy (e.g., surgical treatment, chemotherapy, cryotherapy, radiation therapy, brachytherapy and hormonal therapy).

[0028] In some embodiments, the method further comprises detecting expression of at least one other cancer marker polynucleotide, illustrative examples of which include PCA3 (Prostate Cancer Antigen 3), Claudin 4, Hepsin, PSMA (Prostate Specific Membrane Antigen), SPINKl (Serine Peptidase INhibitor, Kazal type 1), GOLPH2 (GOLgi PHosphoprotein 2), KLK2 (KaLHKrein 2), KLK4 (KaLHKrein 4), KLKIl (KaLHKrein 11), KLKl 4 (KaLHKrein 14), KLKl 5 (KaLHKrein 15), PBOVl (Prostate and Breast cancer OVerexpressed 1) / UROC28 , BCL2 (B-cell CLL/lymphoma 2), TMPRSS2.ERG, GalNAc-T3 (UDP-N-acetyl-alpha-D-GALactosamine:polypeptide N-ACetylgalactosaminylTransferase 3), MUCl (Mucin 1), EGFR, mutant p53, cyclin D, PCNA, Ki67, uPA, PAI (Plasminogen), HER2 (Human Epidermal growth factor Receptor 2) /neu / ERbB2, Cathepsin D, AR (Androgen Receptor), MUCl (MUCinl), EGFR (Epidermal Growth Factor Receptor), mutant p53, cyclin D, PCNA (Proliferating Cell Nuclear Antigen), Ki67, uPA (urokinase type Plaminogen Activator) and PAI (Plaminogen Activator Inhibitor), or an encoded polypeptide product thereof. [0029] The novel upstream sequences broadly described above define four novel exon 1 variants designated exons Ib, Ic, Id and Ie as noted above, which can be spliced together with any one or more of exons 2a, 2b, 2c, 3 and 4. Accordingly, in yet another aspect, the present invention provides isolated polynucleotides that comprise or consists essentially of an exon 1 sequence selected from: (1) an exon Ib sequence as set forth in SEQ ID NO: 2, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 2, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 2 under at least high stringency conditions; (2) an exon Ic sequence as set forth in SEQ ID NO: 3, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 3 or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 3 under at least high stringency conditions; (3) an exon Id sequence as set forth in SEQ ID NO: 4, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 4, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 4 under at least high stringency conditions; and (4) an exon Ie sequence as set forth in SEQ ID NO: 5, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 5, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 5 under at least high stringency conditions. In some embodiments, the polynucleotide further comprises an exon 2a sequence. In some embodiments, the polynucleotide further comprises an exon 2b sequence. In some embodiments, the polynucleotide further comprises an exon 2c sequence. In some embodiments, the polynucleotide further comprises an exon 3 sequence. In some embodiments, the polynucleotide further comprises an exon 4 sequence. In specific embodiments, the polynucleotide further comprises downstream of the exon 1 sequence, an exon 2a sequence, an exon 2b sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2a-exon 2b-exon 2c-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 11-15. In other specific embodiments, the polynucleotide further comprises downstream of the exon 1 sequence, an exon 2a sequence, an exon 2b sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2a-exon 2b-exon 3 -exon 4), illustrative examples of which are set forth in SEQ ID NO: 16-20. In other embodiments, the polynucleotide further comprises downstream of the exon 1 sequence, an exon 2a sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2a-exon 2c-exon 3 -exon 4), illustrative examples of which are set forth in SEQ ID NO: 21-25. In some embodiments, the polynucleotide further comprises downstream of the exon 1 sequence, an exon 2b sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2b-exon 2c-exon 3 -exon 4), illustrative examples of which are set forth in SEQ ID NO: 26-30. In some embodiments, the polynucleotide further comprises downstream of the exon 1 sequence, an exon 2a sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2a-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 31-35. In some embodiments, the polynucleotide further comprises downstream of the exon 1 sequence, an exon 2b sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2b-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 36-40. In some embodiments, the polynucleotide further comprises downstream of the exon 1 sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2c-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 41-45.

[0030] In still another aspect, the present invention provides chimeric nucleic acid constructs comprising an exon 1 polynucleotide as broadly described above, which is operably connected to a regulatory element that is operable in a host cell. In illustrative examples of this type, the constructs are useful, for example, as positive controls in the diagnostic methods of the invention.

[0031] In another aspect, the present invention provides an isolated PC A3 exon 2a polynucleotide that comprises or consists essentially of at least a portion of a nucleic acid sequence selected from SEQ ID NO: 6, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 6 under at least high stringency conditions. Suitably the portion is at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or even at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 or 92 nucleotides in length. [0032] In some embodiments, the polynucleotide further comprises an exon 1 sequence as broadly described above. In some embodiments, the polynucleotide further comprises an exon 2b sequence. In some embodiments, the polynucleotide further comprises an exon 3 sequence. In some embodiments, the polynucleotide further comprises an exon 4 sequence. In specific embodiments, the polynucleotide further comprises an exon 1 sequence, an exon 2b sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked in the order exon 1-exon 2a-exon 2b-exon 2c-exon 3 -exon 4), illustrative examples of which are set forth in SEQ ID NO: 11-15.

[0033] In specific embodiments, the polynucleotide further comprises an exon 1 sequence, an exon 2b sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2a-exon 2b-exon 3 -exon 4), illustrative examples of which are set forth in SEQ ID NO: 16-20. __ __ .

[0034] In other specific embodiments, the polynucleotide further comprises an exon 1 sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1 -exon 2a-exon 2c-exon 3 -exon 4), illustrative examples of which are set forth in SEQ ID NO: 21-25.

[0035] In other specific embodiments, the polynucleotide further comprises an exon 1 sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2a-exon 3-exon 4), illustrative examples of which are set forth in SEQ ID NO: 31-35.

[0036] In still another aspect, the present invention provides chimeric nucleic acid constructs comprising an exon 2a polynucleotide as broadly described above, which is operably connected to a regulatory element that is operable in a host cell. In illustrative examples of this type, the constructs are useful, for example, as positive controls in the diagnostic methods of the invention.

[0037] In another aspect, the present invention provides an isolated PCA3 exon 2b polynucleotide that comprises or consists essentially of at least a portion of a nucleic acid sequence selected from SEQ ID NO: 7, or a sequence that displays at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to the sequence set forth in SEQ ID NO: 7 under at least high stringency conditions. Suitably the portion is at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or even at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 or 92 nucleotides in length. [0038] In some embodiments, the polynucleotide further comprises an exon 1 sequence as broadly described above. In some embodiments, the polynucleotide further comprises an exon 2a sequence. In some embodiments, the polynucleotide further comprises an exon 2c sequence. In some embodiments, the polynucleotide further comprises an exon 3 sequence. In some embodiments, the polynucleotide further comprises an exon 4 sequence. In specific embodiments, the polynucleotide further comprises an exon 1 sequence, an exon 2a sequence, an exon 2c sequence, an exon 3 sequence and a exon 4 sequence (e.g., operably linked in the order exon 1-exon 2a-exon 2b-exon 2c-exon 3 -exon 4), illustrative examples of which are set forth in SEQ ID NO: 11-15. [0039] In specific embodiments, the polynucleotide further comprises an exon 1 sequence, an exon 2a sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked-together in the.order exon 1-exon 2a-exon 2b-exon 3 -exon 4), illustrative examples of which are set forth in SEQ ID NO: 16-20.

[0040] In other specific embodiments, the polynucleotide further comprises an exon 1 sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2b-exon 2c-exon 3 -exon 4), illustrative examples of which are set forth in SEQ ID NO: 26-30.

[0041] In other specific embodiments, the polynucleotide further comprises an exon 1 sequence, an exon 3 sequence and an exon 4 sequence (e.g., operably linked together in the order exon 1-exon 2b-exon 3 -exon 4), illustrative examples of which are set forth in SEQ ID NO: 36-40.

[0042] In still another aspect, the present invention provides chimeric nucleic acid constructs comprising an exon 2b polynucleotide as broadly described above, which is operably connected to a regulatory element that is operable in a host cell. In illustrative examples of this type, the constructs are useful, for example, as positive controls in the diagnostic methods of the invention.

[0043] In still another aspect, the present invention provides isolated host cells containing a nucleic acid construct as broadly described above. In certain embodiments, the host cells are selected from bacterial cells, yeast cells and insect cells. In illustrative examples of this type, the host cells are used in the production of prostate cancer marker polynucleotides for use as positive controls. [0044] In a further aspect, the present invention provides probes for interrogating nucleic acid for the presence of a polynucleotide as broadly described above. These probes generally comprise or consist essentially of a nucleotide sequence that hybridizes under at least low, medium or high stringency conditions to a polynucleotide as broadly described above. In some embodiments, the probes consist essentially of a nucleic acid sequence which corresponds or is complementary to at least a portion of a prostate cancer marker polynucleotide, wherein the portion is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. Representative probes for detecting the prostate cancer marker polynucleotides of the invention are set forth in any one of SEQ ID NO: 46-52.

[0045] In another aspect, the invention provides a kit for diagnosing the presence or _ risk of_deyelopment of prostate cancer and/or metastasis of prostate cancer, comprising one or more probes, as broadly described above. Suitably, in some embodiments, the kit further comprises reagents and instructions for use in the method broadly described above. [0046] In yet another aspect, the invention contemplates use of the methods broadly described above in the monitoring, treatment and management of prostate cancer or prostate cancer metastasis. In these embodiments, the diagnostic/prognostic methods of the invention are typically used at a frequency that is effective to monitor the early development of prostate cancer or to monitor the stage, degree or progression of prostate cancer, to thereby enable early or more effective therapeutic intervention or treatment of the cancer. In illustrative examples, the diagnostic/prognostic methods are used at least at 1-, 2-, 4-, 6-, 8-, 12-, 16- or 18-hour intervals, or at least at 1-, 2-, 3-, 4-, 5- or 6-day intervals, or at least at weekly or monthly intervals.

[0047] In a related aspect, the present invention provides methods for treating or preventing prostate cancer and/or metastasis of prostate cancer in a subject. These methods generally comprise detecting expression or overexpression of a prostate cancer marker polynucleotide in the subject, and administering to the subject at least one therapy that treats or ameliorates the symptoms or reverses or inhibits the development or progression of the prostate cancer in the subject. Representative examples of such therapies include surgery, radiation therapy, chemotherapy, stem cell transplant; hormone therapy, anti-resorptive agent therapy and antibody therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a diagrammatic representation of the partial PC A3 gene structure as originally reported by Bussemakers et al., (1999, Cancer Res, 59: 5975-5979) with 4 exons (open boxes ~ not to scale) with alternate splicing of exon 2 and three alternate transcription termination sites in exon 4.

[0049] Figure 2 is a diagrammatic representation of the structure of the PCA3 transcriptional unit which comprises 6 exons (open boxes ~ not to scale) with alternate splicing of exon 2a, exon 2b and exon 2c (original exon T). Shading identifies the newly identified regions of the PC A3 gene. [0050] Figure 3 is a photographic representation of agarose gels illustrating the results for 5' RACE using prostate tissue sample comprising BPH, prostate cancer and metastatic cancer. The top gel (A) shows positive bands amplified using primer (A) corresponding to SEQ ID NO: 59 (which is complementary to nucleotides 1096 to 1121 of SEQ ID NO: 15). The middle gel (B) shows positive bands amplified using primer (A) corresponding to SEQ ID NO: 59 (which is complementary to nucleotides 1096 to 1121 of SEQ ID NO: 15). The bottom gel (C) show positive bands amplified using primer (B) corresponding to SEQ ID NO: 60 (which is complementary to nucleotides 991 tol014 of SEQ ID NO: 15); primer (C) corresponding to SEQ ID NO: 61 (which is complementary to nucleotides 707 to 729 of SEQ ID NO: 15); and primer (D) corresponding to SEQ ID NO: 62 (which is complementary to nucleotides 427 to 450 of SEQ ID NO: 15). The boxed bands illustrate positive controls generated from the 5' RACE. Bands were excised and cloned into PGEMT and subsequently sequenced to confirm their transcription start site.

[0051] Figure 4 is a photographic representation of an agarose gel illustrating the expression of exon 2a-contanining PCA3 transcripts and exon 2b-containing PC A3 transcripts in tissue from subjects with BPH, prostate cancer and metastasis. The top gel (I) is after RT- PCR amplification of PC A3 from cDNA using exon 1 forward primer (PC A3 -4F) set forth in SEQ ID NO: 46 (nucleotides 1127 to 1150 of SEQ ID NO: 15) and a reverse primer from exon 2c (Ex2cR) set forth in SEQ ID NO: 47 (nucleotides 1558 to 1577 of SEQ ID NO: 15). The lanes represent multiple samples and the higher molecular size fragments were detected only in tissues containing prostate cancer and prostate cancer metastases. The bottom gel (II) is after RT-PCR amplification of PC A3 from cDNA using the exon 1 forward primer (PCA3- 4F) set forth in SEQ ID NO: 46 (nucleotides 1127 to 1150 of SEQ ID NO: 15) and a reverse primer from exon 2a (Ex2aR) set forth in SEQ ID NO: 48 (nucleotides 1325 to 1347 of SEQ ID NO: 15).

[0052] Figure 5 is a photographic representation of an agarose gel illustrating the expression of exon 2c containing PC A3 transcripts in prostate tissue from subjects with BPH, prostate cancer and metastasis. The top gel (I) shows amplified products obtained after RT- PCR amplification and the bottom gel (II) shows amplified products obtained after quantitative PCR. Lane 1 shows an amplified product corresponding to an exon 2c- containing PC A3 transcript amplified by Primer exon 1 Fwd (SEQ ID NO: 49, nucleotides 1156 to 1176 of SEQ ID NO: 15) and primer exon 2c Revs (SEQ ID NO: 50, which is complementary to nucleotides 1558 to 1617 of SEQ ID NO: 15); Lane 2 shows an amplified product corresponding to an exon 2c-containing PCA3 transcript amplified by Primer 1 Fwd (SEQ_ID.NOL51, nucleotides 1127 to 1150 of SEQ ID NO:15).and Primer exon 2c Revs . (SEQ ID NO: 50, which is complementary to nucleotides 1558 to 1617 of SEQ ID NO: 15); Lane 3 shows an amplified product corresponding to the art known PCA3 biomarker identified by Bussemaker et al., 1999, amplified by exon 1 Fwd prior art primer (SEQ ID NO: 53, nucleotides 1492 to 1510 of SEQ ID NO: 15) and exon 4 Rvs prior art primer (SEQ ID NO: 54 which is complementary to nucleotides 1843 to 1861 of SEQ ID NO: 15); Lane 4 shows an amplified product corresponding to the house keeping gene B2M amplified by B2MF (SEQ ID NO: 53) and B2MR (SEQ ID NO: 54); and lane 5 represents the house keeping gene PSA amplified by PSAF (SEQ ID NO: 55) and PSAR (SEQ ID NO: 56).

[0053] Figure 6 is a photographic representation of an agarose gel illustrating the expression of exon 2a and exon 2b-containing PC A3 transcripts in prostate tissue from subjects with BPH, prostate cancer (tumors) and metastasis. The top gel (i) is after 35 cycles of PCR amplification of PC A3 from cDNA using Exon 2a forward primer (Ex2AF) set forth in SEQ ED NO: 64 and a Exon 3 (Ex3R) reverse primer set forth in SEQ ID NO: 65 and the bottom gel (ii) is after 35 cycles of PCR amplification of PC A3 from cDNA using Exon 2b forward primer (Ex2BF) set forth in SEQ ID NO: 66 and Exon 3 reverse primer (Ex3R) set forth hi SEQ ID NO: 65. The lanes represent multiple samples. M represents DNA base pair marker and C represents negative control. [0054] Figure 7 is a graphical representation of an ROC analysis of PC A3 isoform expression hi ejaculate fluids produced from patients who have undergone Trus biopsies. PSA was used to normalised the expression of PCA3 isoforms. The table shows the sensitivity and specificity of the PC A3 isoforms in prostate cancer diagnosis. TABLEA

BRIEF DESCRIPTION OF THE SEQUENCES

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0056] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0057] The term "aberrant expression," as used herein to describe the expression of a prostate cancer polynucleotide, refers to the overexpression of a prostate cancer polynucleotide relative to a 'normal' level of expression of the prostate cancer polynucleotide or allelic variant thereof in healthy or normal cells or in cells obtained from a healthy subject or from a subject lacking prostate cancer, and/or to a level of a prostate cancer polynucleotide in a healthy tissue sample or body fluid or in a tissue sample or body fluid obtained from a healthy subject or from a subject lacking prostate cancer. In particular, a prostate cancer polynucleotide is aberrantly- or over-expressed if the level of expression of the prostate cancer polynucleotide is higher by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or even an at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% than the level of expression of the prostate cancer polynucleotide in healthy or normal cells or in cells obtained from a healthy subject or from a subject without prostate cancer, and/or relative to a level of a prostate cancer polynucleotide in a healthy tissue sample or body fluid or in a tissue sample or body fluid obtained from a healthy subject or from a subject lacking prostate cancer.

[0058] By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0059] "Amplification product" refers to a nucleic acid product generated by nucleic acid amplification techniques. [0060] The term "biological sample" as used herein refers to a sample that may be extracted, untreated, treated, diluted or concentrated from an animal. The biological sample may include cells or cell lines, histological slides, biopsies, paraffin-embedded tissue, bodily fluids, ejaculate, urine, blood, sputum, bone, stool, tissue and prostate tissue. In some embodiments, the biological sample may include a biological fluid such as urine, prostatic fluid, seminal fluid, ejaculate fluid, peripheral blood and the like. In certain embodiments, the biological sample comprises cells from a tissue biopsy such as a prostate biopsy.

[0061] The term "Benign Prostatic Hyperplasia" or "BPH" refers to a medical condition which causes enlargement of the prostate gland, causing the gland to press against the urethra. The condition is not indicative of prostate cancer.

[0062] "Cells," "host cells," "transformed host cells," "regenerable host cells" and the like are terms that not only refer to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0063] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. The phrases "consisting essentially of," "consists essentially of and the like refer to the components which are essential in order to obtain the advantages of the present invention and any other components present would not significantly change the properties related to the inventive concept. Put another way, these phrases refer to the inclusion of any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrases "consisting essentially of," "consists essentially of and the like indicate that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0064] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence "A-G-T," is complementary to the sequence "T-C-A."

Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be "complete" or "total" complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0065] By "corresponds to" or "corresponding to" is meant a polynucleotide having a nucleotide sequence that is ^"substantially identical (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity) to all or a portion of a reference polynucleotide sequence or of a complement thereof. [0066] As used herein the term "cytostatic agent" refers to a substance that can inhibit cell proliferation or cell division without necessarily killing the cell. Suitably, the cytostatic agent inhibits the proliferation of cancer cells.

[0067] The term "cytotoxic agent" or "cytotoxic therapy" as used herein refers to a substance or therapy that is harmful to cells and ultimately causes cell death, hi some embodiments, the cytotoxic agent harms rapidly dividing cells such as cancer cells and causes cancer cell death, especially cancer cell death while not causing damage to or causing less damage to non-cancer cells. An example of a cytotoxic therapy is radiotherapy.

[0068] By "effective amount", in the context of treating or preventing a condition is meant the administration of that amount of active to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. [0069] The term "exon" refers to a nucleic acid sequence in the DNA or RNA transcript following genetic splicing.

[0070] The terms "expression" or "gene expression" refer to either production of RNA message or translation of RNA message into proteins or polypeptides. Detection of either types of gene expression in use of any of the methods described herein are part of the invention, hi specific embodiments, the terms "expression" or "gene expression" refer to transcription of a gene to produce a transcript or RNA message.

[0071] The term "expression vector" is meant a vector or vehicle similar to a cloning vector but which is capable of expressing a gene which has been cloned into it, after transformation into a host. The cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences, such as promoter sequences.

[0072] The term "gene" as used herein refers to any and all discrete coding regions of a host genome, or regions that code for a functional RNA only (e.g., tRNA, rRNA, regulatory RNAs such as ribozymes, post-transcription gene silencing- (PTGS) associated RNAs etc) as well as associated non-coding regions and optionally regulatory regions, hi certain embodiments, the term "gene" includes within its scope the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non-coding nucleotide sequences involved in the regulation of expression, hi this regard, the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals. The gene sequences may be cDNA or genomic DNA or a fragment thereof. The gene may be introduced into an appropriate vector for extra-chromosomal maintenance or for integration into the host.

[0073] The term "host" refers to any organism, or cell thereof, whether eukaryotic or prokaryotic into which a recombinant construct can be stably or transiently introduced.

[0074] "Hybridization" is used herein to denote the pairing of complementary nucleotide sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid. Complementary base sequences are those sequences that are related by the base-pairing rules, hi DNA, A pairs with T and C pairs with G. hi RNA U pairs with A and C pairs with G. In this regard, the terms "match" and "mismatch" as used herein refer to the hybridization potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that do not hybridize efficiently. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances as known to those of skill in the art.

[0075] The phrase "hybridizing specifically to" and the like refer to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

[0076] By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polynucleotide", as used herein, refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment.

[0077] By "metastases" and "metastasis" is meant prostate cancer that has left the prostate gland and its neighbouring organs and has moved to other areas of the body. Advanced prostate cancer bone metastasis and lymph node metastasis, which can be local or distant, are both associated with advanced prostate cancer.

[0078] By "obtained from" is meant that a sample such as, for example, a nucleic acid extract is isolated from, or derived from, a particular source. For instance, the extract may be isolated directly from a biological tissue of the subject.

[0079] The term "oligonucleotide" as used herein refers to a polymer composed of a multiplicity of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogues thereof, including nucleotides with modified or substituted sugar groups and the like) linked via phosphodiester bonds (or related structural variants or synthetic analogues thereof). Thus, while the term "oligonucleotide" typically refers to a nucleotide polymer in which the nucleotide residues and linkages between them are naturally-occurring, it will be understood that the term also includes within its scope various analogues including, but not restricted to, peptide nucleic acids (PNAs), phosphorothioate, phosphorodithioate, phophoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoroamidate, methyl phosphonates, 2-O-methyl ribonucleic acids, and the like. The exact size of the molecule can vary depending on the particular application. Oligonucleotides are a polynucleotide subset with 200 bases or fewer in length. Preferably, oligonucleotides are 10 to 60 bases in length and most preferably 10, 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g., for probes; although oligonucleotides may be double stranded, e.g., for use in the construction of a variant nucleic acid sequence. Oligonucleotides of the invention can be either sense or antisense oligonucleotides.

[0080] By "operably connected" or "operably linked" and the like is meant a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the nucleic acid sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. An exon sequence is "operably linked to" another exon sequence when RNA polymerase will transcribe the two exon sequences into a single mRNA, which is then transcribed into a single transcript with a nucleotide sequence derived from both exon sequences. The coding sequences need not be contiguous to one another so long as the expressed sequences are ultimately processed to produce the desired protein. "Operably connecting" a promoter to a transcribable polynucleotide is meant placing the transcribable polynucleotide (e.g., protein encoding polynucleotide or other transcript) under the regulatory control of a promoter, which then controls the transcription and optionally translation of that polynucleotide. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position a promoter or variant thereof at a distance from the transcription start site of the transcribable polynucleotide, which is approximately the same as the distance between that promoter and the gene it controls in its natural setting; i.e.: the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function. Similarly, the preferred positioning of a regulatory sequence element (e.g., an operator, enhancer etc) with respect to a transcribable polynucleotide to be placed under its control is defined by the positioning of the element in its natural setting; i.e. the genes from which it is derived.

[0081] The term "polynucleotide" or "nucleic acid" as used herein designates mRNA, RNA, cRNA, cDNA or DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non- natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators and modified linkages (e.g., α-anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen binding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. RNA forms of the genetic molecules of the present invention are generally mRNA or iRNA including siRNAs. The genetic form may be in isolated form or integrated with other genetic molecules such as vector molecules and particularly expression vector molecules. The terms "nucleotide sequence," "polynucleotide" and "nucleic acid" used herein interchangeably and encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. The term "polynucleotide" or "nucleic acid" typically refers to oligonucleotides greater than 30 nucleotides in length. [0082] The term "primary prostate cancer" as used herein refers to the original site

(organ or tissue) where the prostate cancer originated from (e.g., the prostate) which is not metastatic.

[0083] By "primer" is meant an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent. The primer is typically single-stranded for maximum efficiency in amplification but may alternatively be double-stranded. A primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the primer may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,- 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, to one base shorter in length than the template sequence at the 3' end of the primer to allow extension of a nucleic acid chain, though the 5' end of the primer may extend in length beyond the 3' end of the template sequence. In certain embodiments, the oligonucleotide primer contains 15 to 35 or more nucleotides, although it may contain fewer nucleotides. Primers can be large polynucleotides, such as from about 200 nucleotides to several kilobases or more. Primers may be selected to be "substantially complementary" to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis. By "substantially complementary," it is meant that the primer is sufficiently complementary to hybridize with a target nucleotide sequence. Suitably, the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential. For example, non-complementary nucleotides may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the template. Alternatively, non-complementary nucleotides or a stretch of non-complementary nucleotides can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer. [0084] "Probe" refers to a molecule that binds to a specific sequence or subsequence or other moiety of another molecule. Unless otherwise indicated, the term "probe" typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labelled directly or indirectly and include primers within their scope. As used herein, the term "probe" encompasses primers which can be used for example in template-dependent nucleic acid extension, ligation or amplification reactions.

[0085] As used herein the term "prognosis" shall be taken to mean a prediction of the progression of the disease (illustrative examples of which include regression, stasis and metastasis), in particular aggressiveness and metastatic potential of a tumour. It is typically used to define patients with high, low and intermediate risks of death or recurrence after treatment that result from the inherent heterogeneity of the disease process. Prognosis may also be referred to in terms of 'aggressiveness' wherein an aggressive cancer is determined to have a high risk of negative outcome and wherein a non-aggressive cancer has a low risk of negative outcome. As used herein the term "aggressive" as used with respect to a tumour shall be taken to mean a cell proliferative disorder that has the biological capability to rapidly spread outside of its primary location or organ. Indicators of tumour aggressiveness standard in the art include but are not limited to tumour stage, tumour grade, Gleason grade, Gleason score, nodal status and survival. As used herein the term "survival" shall not be limited to mean survival until mortality (wherein said mortality may be either irrespective of cause or cell proliferative disorder related) but may be used in combination with other terms to define clinical terms, for example, to "recurrence-free survival" (wherein the term recurrence includes both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease includes cancer and diseases associated therewith). The length of the survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and a defined end point (e.g. death, recurrence or metastasis). [0086] By "promoter" is meant a region of DNA, which controls at least in part the initiation and level of transcription. Reference herein to a "promoter" is to be taken in its broadest context.and includes the. transcriptional regulatory sequences of a classical genomic gene, including a TATA box and CCAAT box sequences, as well as additional regulatory elements (i.e., activating sequences, enhancers and silencers) that alter gene expression in response to developmental and/or environmental stimuli, or in a tissue-specific or cell-type- specific manner. A promoter is usually, but not necessarily, positioned upstream or 5', of a transcribable sequence (e.g., a coding sequence or a sequence encoding a functional RNA), the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene. Promoters according to the invention may contain additional specific regulatory elements, located more distal to the start site to further enhance expression in a cell, and/or to alter the timing or inducibility of expression of a structural gene to which it is operably connected. The term "promoter" also includes within its scope inducible, repressible and constitutive promoters as well as minimal promoters. Minimal promoters typically refer to minimal expression control elements that are capable of initiating transcription of a selected DNA sequence to which they are operably linked. In some examples, a minimal promoter is not capable of initiating transcription in the absence of additional regulatory elements (e.g., enhancers or other cis-acting regulatory elements) above basal levels. A minimal promoter frequently consists of a TATA box or TATA-like box. Numerous minimal promoter sequences are known in the literature. For example, minimal promoters may be selected from a wide variety of known sequences, including promoter regions from fos, CMV, SV40 and IL-2, among many others. Illustrative examples are provided which use a minimal CMV promoter or a minimal IL2 gene promoter (-72 to +45 with respect to the start site; Siebenlist, 1986).

[0087] The term "radiotherapy" as used herein refers to the treatment or exposure of a cancer or cancer cells such as tumour cells to high energy radiation. The effectiveness of radiotherapy may be enhanced by selenate or its pharmaceutically acceptable salt.

Furthermore, radiotherapy may be further enhanced by administration of radiosensitizing agent. Illustrative examples of radiosensitizing agents include but are not limited to efaproxiral, etanidazole, fluosol, misonidazole, nimorazole, temoporfin and tirapazamine.

[0088] The term "secondary prostate cancer" as used herein refers to prostate cancer that has spread (metastasized) from the prostate to other parts of the body (e.g., skeletal tissue, lymph nodes, liver etc).

[0089] The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by an appropriate method. For example, sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. [0090] Terms used to describe sequence relationships between two or more polynucleotides include "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al, 1997, Nucl. Acids Res. 25: 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al, "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15.

[0091] As used herein, the term "stage of cancer" refers to a qualitative or quantitative assessment of the level of advancement of a cancer. Criteria used to determine the stage of a cancer include, but are not limited to, the size of the tumour, whether the tumour has spread to other parts of the body and where the cancer has spread (e.g., within the same organ or region of the body or to another organ).

[0092] "Stringency" as used herein refers to the temperature and ionic strength conditions, and presence or absence of certain organic solvents, during hybridization. The higher the stringency, the higher will be the observed degree of complementarity between sequences. [0093] "Stringent conditions" as used herein refers to temperature and ionic conditions under which only polynucleotides having a high proportion of complementary bases, preferably having exact complementarity, will hybridize. The stringency required is nucleotide sequence dependent and depends upon the various components present during hybridization, and is greatly changed when nucleotide analogues are used. Generally, stringent conditions are selected to be about 10° C to 20° C less than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a target sequence hybridizes to a complementary probe. [0094] The terms "subject" or "individual" or "patient", used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian male subject, for whom therapy or prophylaxis is desired. In specific embodiments, the subject is suspected of having cancer or is at risk of cancer. A preferred subject is a human in need of diagnosis of the presence or absence of prostate cancer and/or metastases. However, it will be understood that the aforementioned terms do not imply that symptoms are present.

[0095] As used herein, the term "subject suspected of having cancer" refers to a subject that presents one or more symptoms indicative of a cancer (e.g., a noticeable lump or mass) or is being screened for a cancer (e.g., during a routine physical). A subject suspected of having cancer may also have one or more risk factors. A subject suspected of having cancer has generally not been tested for cancer. However, a "subject suspected of having cancer" encompasses an individual who has received an initial diagnosis (e.g., a CT scan showing a mass or increased PSA level) but for whom the stage of cancer is not known. The term further includes people who once had cancer (e.g., an individual in remission).

[0096] As used herein, the term "subject at risk for cancer" refers to a subject with one or more risk factors for developing a specific cancer. Risk factors include, but are not limited to, gender, age, genetic predisposition, environmental expose, previous incidents of cancer, pre-existing non-cancer diseases, and lifestyle. [0097] The term "transcribable nucleic acid sequence" or "transcribed nucleic acid sequence" excludes the non-transcribed regulatory sequence that drives transcription. Depending on the aspect of the invention, the transcribable sequence may be derived in whole or in part from any source known to the art, including an animal, eukaryotic, nuclear or plasmid DNA, cDNA, viral DNA or chemically synthesised DNA. A transcribable sequence may contain one or more modifications in either the coding or the untranslated regions, which could affect the biological activity or the chemical structure of the expression product, the rate of expression or the manner of expression control. Such modifications include, but are not limited to, insertions, deletions and substitutions of one or more nucleotides. The transcribable sequence may contain an uninterrupted coding sequence or it may include one or more introns, bound by the appropriate splice junctions. The transcribable sequence may also encode a fusion protein. In other embodiments, the transcribable sequence comprises non- coding regions only. [0098] The term "transcription start site" refers to a distinct region of a DNA molecule that functions as a RNA polymerase binding site and at which point, transcription of the DNA into RNA begins.

[0099] The term "transformation" means alteration of the genotype of a host by the introduction of an expression system according to the invention.

[0100] By "treatment," "treat," "treated" and the like is meant to include both therapeutic and prophylactic treatment.

[0101] The term "upstream" refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. For instance, an upstream nucleotide sequence can be located on the 5' side of a coding sequence or starting point of transcription. In illustrative examples a promoter is located upstream of the start site of transcription and a first exon is located upstream of a second exon.

[0102] The terms "variant" and "polynucleotide variant" refer to polynucleotides displaying substantial sequence identity with a reference polynucleotide sequence or polynucleotides that hybridize with a reference sequence under stringent conditions that are defined hereinafter. These terms also encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains a biological function or activity of the reference polynucleotide. The terms "polynucleotide variant" and "variant" also include naturally-occurring allelic variants.

[0103] By "vector" is meant a nucleic acid molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned. A vector typically contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector may be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a closed circular plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. A vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may also include a marker such as an antibiotic resistance gene that can be used for identification of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.

2. Markers of prostate cancer and uses therefor

[0104] The present invention concerns the detection, diagnosis or prognosis of prostate cancer and/or prostate cancer metastases. Markers of prostate cancer and prostate cancer metastasis, in the form of RNA molecules of specified sequences, of subjects with or susceptible to prostate cancer and metastases, are disclosed. These markers are indicators of prostate cancer and/or prostate cancer metastasis and, when detected or, in some embodiments, when differentially or aberrantly expressed as compared to their expression in normal subjects or in subjects lacking prostate cancer or in subjects with BPH, are diagnostic for the presence of prostate cancer and/or metastasis of prostate cancer in tested subjects. Such markers provide considerable advantages over the prior art in this field. In certain advantageous embodiments, it is possible to diagnose prostate cancer and/or metastasis merely by detecting the presence of a prostate cancer marker polynucleotide of the invention. [0105] In particular, the present inventors have identified two novel exons, exon 2a and exon 2b in the PCA3 gene and unexpectedly found that exon 2a-containing PCA3 transcripts, exon 2b-containing PCA3 transcripts and exon 2c-containing PCA3 transcripts are differentially expressed between biological samples obtained from prostate cancer patients and patients with metastasis of prostate cancer and those obtained from normal subjects and subjects with BPH. Accordingly, in some embodiments, the present invention relates to the use of prostate cancer marker polynucleotides for detection, diagnosis or prognosis of prostate cancer and metastasis of prostate cancer in biological tissue samples, including prostate tissue samples and biological fluid samples, wherein the prostate marker polynucleotides comprise a PCA3 nucleic acid sequence comprising a PC A3 exon 2a (a non-limiting example of which is set forth in SEQ ID NO: 6). In some embodiments, the PCA3 nucleic acid sequence further comprises an exon 1 nucleic acid sequence, non-limiting examples of which include exon Ia, exon Ib, exon Ic, exon Id and exon Ie, as defined herein. Representative examples of exons Ia to Ie are set forth in SEQ ID NO: 1 to 5, respectively. In some embodiments, the PCA3 nucleic acid sequence further comprises an exon 2b nucleic acid sequence. In some embodiments, the PCA3 nucleic acid sequence further comprises an exon 2c nucleic acid sequence. In some embodiments, the PCA3 nucleic acid sequence further comprises an exon 3 nucleic acid sequence. In some embodiments the PCA3 nucleic acid sequence further comprises an exon 4 nucleic acid sequence. Generally, the PCA3 nucleic acid sequence further comprises at least 1, 2, 3, 4, 5 or each of an exon 1 nucleic acid sequence, an exon 2b nucleic acid sequence, an exon 2c nucleic acid sequence, an exon 3 nucleic acid sequence and an exon 4 nucleic acid sequence, non-limiting examples of which are set forth in SEQ ID NO: 11 to 15, respectively. In some embodiments, the PC A3 nucleic acid sequence comprises at least 1, 2, 3, 4 or each of an exon 1 nucleic acid sequence, an exon 2b nucleic acid sequence, an exon 3 nucleic acid sequence and an exon 4 nucleic acid sequence, non-limiting examples of which are set forth in SEQ ID NO: 16 to 20. In some embodiments, the PC A3 nucleic acid sequence further comprises at least 1, 2, 3, 4 or each of an exon 1 nucleic acid sequence, an exon 2c nucleic acid sequence, an exon 3 nucleic acid sequence and an exon 4 nucleic acid sequence, non-limiting examples of which are set forth in SEQ ID NO: 21 to 25. hi some embodiments, the PCA3 nucleic acid sequence further comprises at least 1, 2, 3 or each of an exon 1 nucleic acid sequence, an exon 3 nucleic acid sequence and an exon 4 nucleic acid sequence, non-limiting examples of which are set forth in SEQ ID NO: 31 to 35.

[0106] hi some embodiments, the present invention relates to the use of prostate cancer marker polynucleotides for detection, diagnosis or prognosis of prostate cancer and metastasis of prostate cancer in biological tissue samples, including prostate tissue samples, wherein the prostate marker polynucleotides comprise a PCA3 nucleic acid sequence comprising a PC A3 exon 2b (a non-limiting example of is set forth in SEQ ID NO: 7). In some embodiments, the PCA3 nucleic acid sequence further comprises an exon 1 nucleic acid sequence, non-limiting examples of which include exon Ia, exon Ib, exon Ic, exon Id and exon Ie, as defined herein, hi some embodiments, the PC A3 nucleic acid sequence further comprises an exon 2a nucleic acid sequence. In some embodiments, the PCA3 nucleic acid sequence further comprises an exon 2c nucleic acid sequence. In some embodiments, the PCA3 nucleic acid sequence further comprises an exon 3 nucleic acid sequence. In some embodiments the PC A3 nucleic acid sequence further comprises an exon 4 nucleic acid sequence. Generally, the PCA3 nucleic acid sequence further comprises at least 1, 2, 3, 4, 5 or each of an exon 1 nucleic acid sequence, an exon 2a nucleic acid sequence, an exon 2c nucleic acid sequence, an exon 3 nucleic acid sequence and an exon 4 nucleic acid sequence, non- limiting examples of which are set forth in SEQ ID NO: 11 to 15. In some embodiments, the PC A3 nucleic acid sequence further comprises 1, 2, 3, 4 or each of an exon 1 nucleic acid sequence, an exon 2a nucleic acid sequence, an exon 3 nucleic acid sequence and an exon 4 nucleic acid sequence, non-limiting examples of which are set forth in SEQ ID NO: 16 to 20. In some embodiments, the PC A3 nucleic acid sequence further comprises at least 1, 2, 3, 4 or each of an exon 1 nucleic acid sequence, an exon 2c nucleic acid sequence, an exon 3 nucleic acid sequence and an exon 4 nucleic acid sequence, non-limiting examples of which are set forth in SEQ ID NO: 26 to 30. In some embodiments, the PC A3 nucleic acid sequence further comprises at least 1, 2, 3 or each of an exon 1 nucleic acid sequence, an exon 3 nucleic acid sequence and an exon 4 nucleic acid sequence, non-limiting examples of which are set forth in SEQ ID NO: 36 to 40. [0107] . - In some embodiments, the present invention relates to the use of prostate cancer marker polynucleotides for detection, diagnosis or prognosis of prostate cancer and metastasis of prostate cancer in biological tissue samples, including prostate tissue samples, wherein the prostate marker polynucleotides comprise a PC A3 nucleic acid sequence comprising a PCA3 exon 2c (a non-limiting example of which is set forth in SEQ ID NO: 8). In some embodiments, the PCA3 nucleic acid sequence further comprises an exon 1 nucleic acid sequence, non-limiting examples of which include exon Ia, exon Ib, exon Ic, exon Id and exon Ie, as defined herein. In some embodiments, the PC A3 nucleic acid sequence further comprises an exon 2a nucleic acid sequence, hi some embodiments, the PCA3 nucleic acid sequence further comprises an exon 2b nucleic acid sequence. In some embodiments, the PCA3 nucleic acid sequence further comprises an exon 3 nucleic acid sequence. In some embodiments the PCA3 nucleic acid sequence further comprises an exon 4 nucleic acid sequence. Generally, the PCA3 nucleic acid sequence further comprises at least 1, 2, 3, 4, 5 or each of an exon 1 nucleic acid sequence, an exon 2a nucleic acid sequence, an exon 2b nucleic acid sequence, an exon 3 nucleic acid sequence and an exon 4 nucleic acid sequence, non-limiting examples of which are set forth in SEQ ID NO: 11 to 15. In some embodiments, the PC A3 nucleic acid sequence further comprises 1, 2, 3, 4 or each of an exon 1 nucleic acid sequence, an exon 2a nucleic acid sequence, an exon 3 nucleic acid sequence and an exon 4 nucleic acid sequence, non-limiting examples of which are set forth in SEQ ID NO: 21 to 25. In some embodiments, the PC A3 nucleic acid sequence further comprises at least 1, 2, 3, 4 or each of an exon 1 nucleic acid sequence, an exon 2b nucleic acid sequence, an exon 3 nucleic acid sequence and an exon 4 nucleic acid sequence, non-limiting examples of which are set forth in SEQ ID NO: 26 to 30. In some embodiments, the PCA3 nucleic acid sequence further comprises at least 1, 2, 3 or each of an exon 1 nucleic acid sequence, an exon 3 nucleic acid sequence and an exon 4 nucleic acid sequence, non-limiting examples of which are set forth in SEQ ID NO: 41 to 45. [0108] The present inventors have also identified four novel transcription start sites upstream of the archetypal transcription start site of the PC A3 gene, which is located at nucleotide 1151 of the sequence set forth in SEQ ID NO: 15. The novel transcription start sites, which are located at nucleotides 1 (also referred to herein as PC A3 -3), 452 (also referred to herein as PCA3-4), 511 (also referred to herein as PCA3-5) and 1015 (also referred to herein as PC A3 -6) of the sequence set forth in SEQ ID NO: 15, extend the size of the prototypic PCA3 exon Ia by 1150, 699, 640 and 136 nucleotides, respectively, and define four.no.vel exon l variants as follows: .

[0109] (1) a 256-nucleotide exon Ib, which includes nucleotides 1015 to 1270 of the sequence set forth in SEQ ID NO: 15, and which is set forth in SEQ ID NO: 2; [0110] (2) a 760-nucleotide exon Ic, which includes nucleotides 511 to 1270 of the sequence set forth in SEQ ID NO: 15, and which is set forth in SEQ ED NO: 3;

[0111] (3) an 819-nucleotide exon Id, which includes nucleotides 452 to 1270 of the sequence set forth in SEQ ID NO: 15, and which is set forth in SEQ ID NO: 4; and

[0112] (4) a 1270-nucleotide exon Ie, which includes nucleotides 1 to 1270 of the sequence set forth in SEQ ID NO: 15, and which is set forth in SEQ ID NO: 5.

[0113] The novel transcription start sites also define novel sequences upstream of the archetypal PC A3 transcription start site, as set forth, for example, in SEQ ID NO: 6, 7, 8 9 and 10, which are useful for example in detecting the prostate cancer marker polynucleotides on the present invention. [0114] Accordingly, it will be apparent that the nucleic acid sequences disclosed herein will find utility in a variety of applications in detection, diagnosis and prognosis of prostate cancer and metastasis of prostate cancer. Examples of such applications within the scope of the present disclosure comprise amplification of prostate cancer marker polynucleotides using specific primers, detection of prostate cancer marker polynucleotides by hybridization with oligonucleotide probes, incorporation of isolated nucleic acids into vectors, expression of vector-incorporated nucleic acids as RNA and protein, and development of immunological reagents corresponding to marker encoded products. [0115] The identified prostate cancer marker polynucleotides may in turn be used to design specific oligonucleotide probes and primers. Such probes and primers may be of any length that would specifically hybridize to the identified marker sequences and may be at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 5 50, 75, 100, 150, 200, 300, 400, 500 nucleotides in length and in the case of probes, up to the full length of the sequences of the prostate cancer marker polynucleotides identified herein. Probes may also include additional sequence at their 5' and/or 3' ends so that they extent beyond the target sequence with which they hybridize.

[0116] When used in combination with nucleic acid amplification procedures, these0 probes and primers enable the rapid analysis of biological samples for detecting or quantifying prostate cancer marker polynucleotides. Such procedures include any method or __. technique kno-wn.in.the art or described herein for duplicating or increasing.the number of . copies or amount of a target nucleic acid or its complement. Generally, the biological sample may contain cells from a tissue biopsy (e.g., prostatectomy specimen or TRUS biopsy) or may5 comprises a biological fluid (e.g., urine, prostatic excretion, prostatic fluid and/or ejaculate fluid). The urine may be collected pre or post ejaculation and the prostatic fluid may be collected after digital rectal examination (DRE).

[0117] One of ordinary skill in the art can select segments from the identified marker polynucleotides for use in the different detection, diagnostic, or prognostic methods, vector constructs, kit, and/or any of the embodiments described herein as part of the present invention. Illustrative prostate cancer marker polynucleotides sequences that are desirable for use in the present invention are those set forth in SEQ ID NO: 11-45 (see Table A).

[0118] As described in the Examples and in the brief description of the sequences, the present disclosure provides prostate cancer marker polynucleotides specifically expressed or aberrantly expressed in subjects with prostate cancer and/or metastasis of prostate cancer or at risk of developing prostate cancer, hi accordance with the present invention, the polynucleotide sequences disclosed herein find utility inter alia as hybridization probes or amplification primers. These nucleic acids may be used, for example, in diagnostic evaluation of biological samples or employed to clone full-length cDNAs or genomic clones corresponding thereto. In certain embodiments, these probes and primers represent oligonucleotides, which are of sufficient length to provide specific hybridization to a RNA or DNA sample extracted from the biological sample. The sequences typically will be about 10- 20 nucleotides, but may be longer. Longer sequences, e.g., of about 30, 40, 50, 100, 500 and even up to full-length, are desirable for certain embodiments.

[0119] Nucleic acid molecules having contiguous stretches of about 10, 15, 17, 20, 30, 40, 50, 60, 75 or 100 or 500 nucleotides of a sequence set forth in SEQ ID NO: 6, 7 and 8, are contemplated. Molecules that are complementary to the above mentioned sequence and that bind to this sequence under high stringency conditions are also contemplated. These probes are useful in a variety of hybridization embodiments, such as Southern and northern blotting, hi some cases, it is contemplated that probes may be used that hybridise to multiple target sequences without compromising their ability to effectively diagnose prostate cancer and/or metastases. In general, it is contemplated that the hybridization probes described herein are useful both as reagents in solution hybridization, as in PCR, for detection of expression of cQrresponding.genes,.asΛvell.as.in embodiments employing a.solid phase .

[0120] Various probes and primers may be designed around the disclosed nucleotide sequences. For example, in certain embodiments, the sequences used to design probes and primers may include repetitive stretches of adenine nucleotides (poly- A tails) normally attached at the ends of the RNA for the identified prostate cancer transcripts. In other embodiments, probes and primers may be specifically designed to not include these or other segments from the identified prostate cancer marker polynucleotide sequences, as one of ordinary skill in the art may deem certain segments more suitable for use in the detection methods disclosed. In any event, the choice of primer or probe sequences for a selected application is within the realm of the ordinary skilled practitioner. Illustrative probe sequences for the detection of prostate cancer marker polynucleotides are listed in Table A (e.g., SEQ ID NO: 46-52).

[0121] Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is desirable. Probes, while perhaps capable of priming, are designed to bind to a target DNA or RNA and need not be used in an amplification process. In certain embodiments, the probes or primers are labelled with radioactive species 32P, 14C, 35S, 3H, or other label), with a fluorophore (e.g., rhodamine, fluorescein) or with a chemillumiscent label (e.g., luciferase). [0122] The present invention provides substantially full-length cDNA sequences as well as EST and partial cDNA sequences that are useful as markers of prostate cancer. It will be understood, however, that the present disclosure is not limited to these disclosed sequences and is intended particularly to encompass at least isolated nucleic acids that are hybridisable to nucleic acids comprising the disclosed sequences or that are variants of these nucleic acids. For example, a nucleic acid of partial sequence may be used to identify a structurally-related gene or the full-length genomic or cDNA clone from which it is derived. Methods for generating cDNA and genomic libraries which may be used as a target for the above- described probes are known in the art (see, for example, Sambrook et al, 1989, supra and Ausubel et al, 1994, supra). Additionally, the present invention includes within its scope isolated or purified expression products of prostate cancer marker polynucleotides (i.e., RNA transcripts).

[0123] Accordingly, the present invention encompasses isolated or substantially purified nucleic acid compositions. An "isolated" or "purified" nucleic acid molecule or protein, or portion thereof, is substantially or essentially free from components that normally accompany orinteract with the Jiucleic acid molecule, as-foiind in its-naturally Occurring environment. Thus, an isolated or purified polynucleotide is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesised. Suitably, an

"isolated" polynucleotide is free of sequences (especially protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide was derived. For example, in various embodiments, an isolated prostate cancer marker polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide was derived.

[0124] The present invention also encompasses portions of the full-length or substantially full-length nucleotide sequences of the prostate cancer marker polynucleotides or their transcripts or DNA copies of these transcripts. Portions of prostate cancer marker polynucleotide sequences may range from at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or even at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or even at least 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or even at least 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or 5100 nucleotides in length, or almost up to the full-length nucleotide sequence.

[0125] The invention also contemplates variants of the prostate cancer marker polynucleotide sequences. Nucleic acid variants can be naturally-occurring, such as allelic variants (same locus), homologies (different locus), and orthologs (different organism) or can be non naturally-occurring. Naturally occurring variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridisation techniques as known in the art. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. Generally, variants of a particular nucleotide sequence of the invention will have at least about 30%, 40% 50%, 55%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, desirably about 90% to 95% or more, and more suitably about 96%, 97%, 98% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.

[0126] The prostatexancer-marker polynucleotide.sequences~of the invention can be used to isolate corresponding sequences and alleles from other organisms, particularly other mammals. Methods are readily available in the art for the hybridization of nucleic acid sequences. Coding sequences from other organisms may be isolated according to well known techniques based on their sequence identity with the coding sequences set forth herein. In these techniques all or part of the known coding sequence is used as a probe which selectively hybridizes to other prostate cancer marker coding sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. Accordingly, the present invention also contemplates polynucleotides that hybridise to the prostate cancer marker polynucleotide sequences, or to their complements (which may be full-length or particle length complements), under stringency conditions described below. As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Ausubel et al, (1998, supra), Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used. Reference herein to low stringency conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C, and at least about 1 M to at least about 2 M salt for washing at 42° C. Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at room temperature. One embodiment of low stringency conditions includes hybridization in 6 x sodium chloride/sodium citrate (SSC) at about 45.° C, followed by two washes in 0.2 x SSC, 0.1% SDS at least at 50° C (the temperature of the washes can be increased to 55° C for low stringency conditions). Medium stringency conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C, and at least about 0.1 M to at least about 0.2 M salt for washing at 55° C. Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS for washing at 60-65° C. One embodiment of medium stringency conditions includes hybridising in 6 x SSC at about 45.° C, followed by one or more washes in 0.2 x SSC, 0.1% SDS. at_60_°. C Jffigh_stringency__conditions include and..encompassirQm_at ieastaboutJ ±%jy/y _ to at least about 50% v/v formamide and from about 0.01 M to about 0.15 M salt for hybridization at 42° C, and about 0.01 M to about 0.02 M salt for washing at 55° C. High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C, and (i) 0.2 x SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C. One embodiment of high stringency conditions includes hybridising in 6 x SSC at about 45.°C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 65° C. [0127] In certain embodiments, a prostate cancer marker polynucleotide is one that hybridizes to a disclosed nucleotide sequence under very high stringency conditions. One embodiment of very high stringency conditions includes hybridising 0.5 M sodium phosphate, 7% SDS at 65° C, followed by one or more washes at 0.2 x SSC, 1% SDS at 65° C.

[0128] Other stringency conditions are well known in the art and a skilled addressee will recognise that various factors can be manipulated to optimise the specificity of the hybridization. Optimisation of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see Ausubel et al, supra at pages 2.10.1 to 2.10.16 and Sambrook et al, (1989, supra) at sections 1.101 to 1.104.

[0129] While stringent washes are typically carried out at temperatures from about 42° C to 68° C, one skilled in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridization rate typically occurs at about 20° C to 25° C below the Tm for formation of a DNA-DNA hybrid. It is well known in the art that the Tm is the melting temperature, or temperature at which two complementary polynucleotide sequences dissociate. Methods for estimating Tm are well known in the art (see Ausubel et al., supra at page 2.10.8). In general, the Tm of a perfectly matched duplex of DNA may be predicted as an approximation by the formula: [0130] Tm = 81.5 + 16.6 (loglO M) + 0.41 (%G+C) - 0.63 (% formamide) -

(600/length).

[0131] wherein: M is the concentration of Na+, preferably in the range of 0.01 molar to 0.4 molar; %G+C is the sum of guanosine and cytosine bases as a percentage of the total number of bases, within the range between 30% and 75% G+C; % formamide is the percent formamide concentration by volume; length is the number of base pairs in the DNA duplex. The Tm of a duplex DNA decreases by approximately 1° C with every increase of 1% in theΗumber of ϊandomly mismatched base paks.^"Wasning^"is^~g^"enerally^~cafried out at TnT- ^" 15° C for high stringency, or Tm - 30° C for moderate stringency.

[0132] In one example of a hybridization procedure, a membrane (e.g., a nitrocellulose membrane or a nylon membrane) containing immobilised DNA is hybridised overnight at 42° C in a hybridization buffer (50% deionised formamide, 5 x SSC, 5 x Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/mL denatured salmon sperm DNA) containing labelled probe. The membrane is then subjected to two sequential medium stringency washes (i.e., 2 x SSC, 0.1% SDS for 15 min at 45° C, followed by 2 x SSC, 0.1% SDS for 15 min at 50° C), followed by two sequential higher stringency washes (i.e., 0.2 x SSC, 0.1% SDS for 12 min at 55° C followed by 0.2 x SSC and 0.1%SDS solution for 12 min at 65-68° C.

3. Methods of detecting prostate cancer marker polynucleotides

[0133] The present invention is predicated in part on the discovery that the presence or risk of prostate cancer and/or prostate cancer metastasis is diagnosed by detecting the presence of a prostate cancer marker in biological samples comprising certain tissue, cells or fluid. In some embodiments, the presence or risk of development of prostate cancer and/or metastases is diagnosed merely by detecting the presence of a prostate cancer marker in a biological sample from a tested subject. In other embodiments, the presence or risk of development of prostate cancer and/or metastases is diagnosed when a prostate cancer marker is detectable at a higher level in a biological sample as compared to the level at which that prostate cancer marker is detected in a reference sample obtained from normal subjects or from subjects lacking that condition. Generally, such diagnoses are made when the level of a prostate cancer marker polynucleotide in the biological sample varies from the level of a corresponding prostate cancer marker polynucleotide in the reference sample by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99%, or even at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999%, or even by at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%. In some embodiments, the absence of prostate cancer and/or metastases is diagnosed when the measured level of a prostate cancer marker polynucleotide of the invention is the same as or similar to the measured level of the corresponding prostate cancer marker polynucleotide in a reference sample obtained from normal subjects or from subjects lacking prostate cancer and/or metastases or from subjects with BPH. In these embodiments, the measured level of a prostate cancer marker polynucleotide varies from the measured level or functional activity of a corresponding prostate cancer marker polynucleotide, by no more than about 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.1%. The corresponding prostate cancer marker polynucleotide is generally selected from the same prostate cancer marker polynucleotide that is present in the biological sample, or a gene product expressed from a variant prostate cancer marker polynucleotide (e.g., a homologous transcript) including a splice variant. In some embodiments, these sequences provide considerable advantage over the prior art in this field, in some embodiments, and are capable of distinguishing between prostate cancer and/or prostate cancer metastasis and BPH in a biological sample from a mammalian male.

[0134] Generally the biological sample contains cells from a tissue biopsy (e.g., prostatectomy specimens and TRUS biopsies) or may contain a biological fluid (e.g., urine, ejaculate fluid and/or prostatic fluid and peripheral blood). [0135] Nucleic acids used in polynucleotide-based assays can be isolated from cells and/or fluid contained in a biological sample, according to standard methodologies (Sambrook, et ah, 1989, supra; and Ausubel et al, 1994, supra). The nucleic acid is typically fractionated (e.g., poly A+ RNA) or whole cell RNA. Where RNA is used as the subject of detection, it may be desired to convert the RNA to a complementary DNA. In some embodiments, the nucleic acid is amplified by a template-dependent nucleic acid amplification technique. A number of template dependent processes are available to amplify at least a portion of a prostate cancer marker polynucleotide present in a given template sample. An exemplary nucleic acid amplification technique is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, Ausubel et al, (supra), and in Innis et al, ("PCR Protocols", Academic Press, Inc., San Diego Calif., 1990). Briefly, in PCR, two primer sequences are prepared that are complementary to regions on opposite complementary strands of a polynucleotide sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If a cognate polynucleotide sequence is present in a sample, the primers will bind to the polynucleotide and the polymerase will cause the primers to be extended along the sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the polynucleotide sequence to form reaction products, excess primers will bind to the sequence and to the reaction products and the process is repeated. A reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989, supra. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art.

[0136] In certain embodiments, the template-dependent amplification involves the quantification of nucleic acid molecules in real-time. For example, RNA or DNA may be quantified using the Real-Time PCR technique (Higuchi, 1992, et al, Biotechnology, 10:413- 417). By determining the concentration of the amplified products of a target DNA sequence in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesised from RNAs isolated from different tissues or cells, the relative abundance of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundance is only true in the linear range of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. [0137] Another method for amplification is the ligase chain reaction ("LCR"), disclosed in EPO No. 320 308. hi LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.

[0138] Qβ Replicase, described in PCT Application No. PCT/US87/00880, may also be used. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected.

[0139] An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'α- thio-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention, Walker et al, (1992, Proc. Natl. Acad. Sci. U.S.A, 89: 392-396).

[0140] Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids, which involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Specific target sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3' and 5' sequences of non-specific DNA and a middle sequence of specific RNA is hybridised to DNA that is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. [0141] Still another amplification method described in GB Application No. 2 202

328, and in PCT Application No. PCT/US89/01025, may be used. In the former application, "modified" primers are used in a PCR-like, template- and enzyme-dependent synthesis. The primers may be modified by labelling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labelled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labelled probe signals the presence of the target sequence. [0142] Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 1173; Gingeras et al, PCT Application WO 88/10315). In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization. The double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.

[0143] Davey et al, EPO No. 329 822 disclose a nucleic acid amplification process involving cyclically synthesising single-stranded RNA ("ssRNA"), ssDNA, and double- stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA. [0144] Miller et al. , in PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include "RACE" and "one-sided PCR" (Frohman, M. A., In: "PCR Protocols: A Guide to Methods and Applications", Academic Press, N. Y., 1990; Ohara et al, 1989, Proc. Natl Acad. Sci. U.S.A., 86: 5673-567).

[0145] Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, may also be used for amplifying target nucleic acid sequences. Wu et al, (1989, Genomics, 4: 560).

[0146] Depending on the format, at least a portion of the prostate cancer marker nucleic acid sequence is identified in the sample directly using a template-dependent amplification as described, for example, above, or with a second, known nucleic acid following amplification. Next, the identified product is detected. In certain applications, the detection may be performed by visual means (e.g., ethidium bromide staining of a gel). Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Afrymax Technology; Bellus, 1994, J Macromol. Sci. Pure, Appl. Chem., A31(l): 1355-1376).

[0147] In some embodiments, amplification products or "amplicons" are visualised in order to confirm amplification of the at least a portion of the prostate cancer marker nucleic acid sequence of interest. One typical visualisation method involves staining of a gel with ethidium bromide and visualisation under UV light. Alternatively, if the amplification products are integrally labelled with radio- or fluorometrically-labelled nucleotides, the amplification products can then be exposed to x-ray film or visualised under the appropriate stimulating spectra, following separation. In some embodiments, visualisation is achieved indirectly. Following separation of amplification products, a labelled nucleic acid probe is brought into contact with the amplified prostate cancer marker sequence. The probe is suitably conjugated to a chromophore but may be radiolabeled. Alternatively, the probe is conjugated to a binding partner, such as an antigen-binding molecule, or biotin, and the other member of the binding pair carries a detectable moiety or reporter molecule. The techniques involved are well known to those of skill in the art and can be found in many standard texts on molecular protocols (e.g., see Sambrook et al, 1989, supra and Ausubel et al 1994, supra). For example, chromophore or radiolabel probes or primers identify the target sequence during or following amplification.

[0148] In certain embodiments, target nucleic acids are quantified using blotting techniques, which are well known to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provide different types of information, although cDNA blotting is analogous, in many aspects, to blotting or RNA species. Briefly, a probe is used to target a DNA or RNA species that has been immobilised on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter. Subsequently, .the .blotted, target is. incubated with.a probe (usually labelled) under conditions that promote denaturation and re-hybridization. Because the probe is designed to base pair with the target, the probe will bind a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above.

[0149] Following detection/quantification, one may compare the results seen in a given subject with a control reaction or a statistically significant reference group of normal subjects or of subjects lacking prostate cancer and/or prostate cancer metastasis. In this way, it is possible to correlate the amount of at least a portion of a prostate cancer marker nucleic acid detected with the presence of the disease.

[0150] Also contemplated are genotyping methods and allelic discrimination methods and technologies such as those described by Kristensen et al, (Biotechniques, 30(2): 318-322), including the use of single nucleotide polymorphism analysis, high performance liquid chromatography, TaqMan®, liquid chromatography, and mass spectrometry. [0151] The present invention also contemplates biochip-based technologies such as those described by Hacia et al, (1996, Nature Genetics 14: 441-447) and Shoemaker et al, (1996, Nature Genetics, 14: 450-456). Briefly, these techniques involve quantitative methods for analysing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ biochip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al, (1994, Proc. Natl. Acad. Sci. U.S.A. 91: 5022-5026); Fodor et al (1991, Science, 251 : 767-773). Briefly, nucleic acid probes to at least a portion of a prostate cancer marker polynucleotide are made and attached to biochips to be used in screening and diagnostic methods, as outlined herein. The nucleic acid probes attached to the biochip are designed to be substantially complementary to specific expressed prostate cancer marker polynucleotides, i.e., a target sequence (either a target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs. This complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the nucleic acid probes of the present invention. However, if the number of mismatches is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. In certain embodiments, more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being desirable, are used to build in a redundancy for a particular target. The probes can be overlapping (i.e. have some sequence in common), or separate. [0152] As will be appreciated by those of ordinary skill in the art, nucleic acids can be attached to or immobilised on a solid support in a wide variety of ways. By "immobilized" and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below. The binding can be covalent or non- covalent. By "non-covalent binding" and grammatical equivalents herein is meant one or more of either, electrostatic, hydrophilic, and hydrophobic interactions. Included in non- covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin. By "covalent binding" and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilisation may also involve a combination of covalent and non-covalent interactions. [0153] In general, the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art. As described herein, the nucleic acids can either be synthesised first, with subsequent attachment to the biochip, or can be directly synthesised on the biochip. [0154] The biochip comprises a suitable solid or semi-solid substrate or solid support. By "substrate" or "solid support" is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method. As will be appreciated by practitioners in the art, the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalised glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon®, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc. In general, the substrates allow optical detection and do not appreciably fluorescese.

. [0155] Generally the substrate is planar, although as will be appreciated by those of skill in the art, other configurations of substrates may be used as well. For example, the probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimise sample volume. Similarly, the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.

[0156] In certain embodiments, oligonucleotides probes are synthesised on the substrate, as is known in the art. For example, photoactivation techniques utilising photopolymerisation compounds and techniques can be used. In an illustrative example, the nucleic acids are synthesised in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505; U.S. Pat. Nos. 5,700,637 and 5,445,934; and references cited within; these methods of attachment form the basis of the Affymetrix GeneChip® technology.

[0157] In an illustrative biochip analysis, oligonucleotide probes on the biochip are exposed to or contacted with a nucleic acid sample suspected of containing one or more prostate cancer marker polynucleotides under conditions favouring specific hybridization. Sample extracts of DNA or RNA, either single or double-stranded, may be prepared from fluid suspensions of biological materials, or by grinding biological materials, or following a cell lysis step which includes, but is not limited to, lysis effected by treatment with SDS (or other detergents), osmotic shock, guanidinium isothiocyanate and lysozyme. Suitable DNA, which may be used in the method of the invention, includes cDNA. Such DNA may be prepared by any one of a number of commonly used protocols as for example described in Ausubel, et al, 199 >4, supra, and Sambrook, et al., 19S9, supra. [0158] Suitable RNA, which may be used in the method of the invention, includes messenger RNA, complementary RNA transcribed from DNA (cRNA) or genomic or subgenomic RNA. Such RNA may be prepared using standard protocols as for example described in the relevant sections of Ausubel, et al. 1994, supra and Sambrook, et al. 1989, supra).

[0159] cDNA may be fragmented, for example, by sonication or by treatment with restriction endonucleases. Suitably, cDNA is fragmented such that resultant DNA fragments are of a length greater than the length of the immobilised oligonucleotide probe(s) but small enough to allow rapid access thereto under suitable hybridization conditions. Alternatively, fragments of cDNA may be selected and amplified using a suitable nucleotide amplification technique, as described for example above, involving appropriate random or specific primers.

[0160] ^* Usualiy^~at least ^"a^pportion of a prostate cancer marker polynucleotide is^" detectably labelled so that its hybridization to a probe can be determined. Polynucleotides are typically detectably labelled with a reporter molecule illustrative examples of which include chromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, bioluminescent molecules, lanthanide ions (e.g., Eu34), a radioisotope and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non- metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like. Illustrative labels of this type include large colloids, for example, metal colloids such as those from gold, selenium, silver, tin and titanium oxide. In some embodiments in which an enzyme is used as a direct visual label, biotinylated bases are incorporated into a target polynucleotide. Hybridization is detected by incubation with streptavidin-reporter molecules.

[0161] Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin

(RPE), and Texas Red. Other exemplary fluorochromes include those discussed by Dower et al, (International Publication WO 93/06121). Reference also may be made to the fluorochromes described in U.S. Patents 5,573,909 (Singer et al), 5,326,692 (Brinkley et al). Alternatively, reference may be made to the fluorochromes described in U.S. Patent Nos. 5,227,487, 5,274,113, 5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517, 5,459,276, 5,516,864, 5,648,270 and 5,723,218. Commercially available fluorescent labels include, for example, fluorescein phosphoramidites such as Fluoreprime® (Pharmacia), Fluoredite® (Millipore) and FAM (Applied Biosystems International) [0162] Radioactive reporter molecules include, for example, 32P, which can be detected by an X-ray or phosphorimager techniques.

[0163] The hybrid-forming step can be performed under suitable conditions for hybridising oligonucleotide probes to test nucleic acid including DNA or RNA. In this regard, reference may be made, for example, to Nucleic Acid Hybridization, A Practical Approach (Homes and Higgins, Eds.) (IRL press, Washington D.C., 1985). In general, whether hybridization takes place is influenced by the length of the oligonucleotide probe and the polynucleotide sequence under test, the pH, the temperature, the concentration of mono- and divalent cations, the proportion of G and C nucleotides in the hybrid-forming region, the viscosity of the medium and the possible presence of denaturants. Such variables also influence the time required for hybridization. The preferred conditions will therefore depend upon the particular application. Such empirical conditions, however, can.be routinely determined without undue experimentation.

[0164] In certain advantageous embodiments, high discrimination hybridization conditions are used. For example, reference may be made to Wallace et al, (1979, Nucl. Acids Res. 6: 3543) who describe conditions that differentiate the hybridisation of 11 to 17 base long oligonucleotide probes that match perfectly and are completely homologous to a target sequence as compared to similar oligonucleotide probes that contain a single internal base pair mismatch. Reference also may be made to Wood et al, (1985, Proc. Natl. Acid. Sci. USA, 82: 1585) who describe conditions for hybridization of 11 to 20 base long oligonucleotides using 3 M tetramethyl ammonium chloride wherein the melting point of the hybrid depends only on the length of the oligonucleotide probe, regardless of its GC content. In addition, Drmanac et al, (supra) describe hybridization conditions that allow stringent hybridization of 6-10 nucleotide long oligomers, and similar conditions may be obtained most readily by using nucleotide analogues such as 'locked nucleic acids (Christensen et al, 2001 Biochem J, 354: 481-4).

[0165] Generally, a hybridization reaction can be performed in the presence of a hybridization buffer that optionally includes a hybridization optimising agent, such as an isostabilizing agent, a denaturing agent and/or a renaturation accelerant. Examples of isostabilizing agents include, but are not restricted to, betaines and lower tetraalkyl ammonium salts. Denaturing agents are compositions that lower the melting temperature of double stranded nucleic acid molecules by interfering with hydrogen bonding between bases in a double stranded nucleic acid or the hydration of nucleic acid molecules. Denaturing agents include, but are not restricted to, formamide, formaldehyde, dimethylsulphoxide, tetraethyl acetate, urea, guanidium isothiocyanate, glycerol and chaotropic salts. Hybridization accelerants include heterogeneous nuclear ribonucleoprotein (hnRP) Al and cationic detergents such as cetyltrimethylammonium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB), polylysine, spermine, spermidine, single stranded binding protein (SSB), phage T4 gene 32 protein and a mixture of ammonium acetate and ethanol. Hybridization buffers may include target polynucleotides at a concentration between about 0.005 nM and about 50 nM, preferably between about 0.5 nM and 5 nM, more preferably between about 1 nM and 2 nM. [0166] A hybridization mixture containing at least a portion of a prostate cancer polynucleotide is placed in contact with the array of probes and incubated at a temperature and for.ajtime appropriate, to permit hybridization between. the target sequences, in the target _ polynucleotides and any complementary probes. Contact can take place in any suitable container, for example, a dish or a cell designed to hold the solid support on which the probes are bound. Generally, incubation will be at temperatures normally used for hybridization of nucleic acids, for example, between about 20° C and about 75° C, example, about 25° C, about 30° C, about 35° C, about 40° C, about 45° C, about 50° C, about 55° C, about 60° C, or about 65° C. For probes longer than 14 nucleotides, 20° C to 50° C is desirable. For shorter probes, lower temperatures are preferred. A sample of target polynucleotides is incubated with the probes for a time sufficient to allow the desired level of hybridization between the target sequences in the target polynucleotides and any complementary probes. For example, the hybridization may be carried out at about 45° C +/-10° C in formamide for 1-2 days.

[0167] After the hybrid-forming step, the probes are washed to remove any unbound nucleic acid with a hybridization buffer, which can typically comprise a hybridization optimising agent in the same range of concentrations as for the hybridization step. This washing step leaves only bound target polynucleotides. The probes are then examined to identify which probes have hybridised to a target polynucleotide.

[0168] The hybridization reactions are then detected to determine which of the probes has hybridised to a corresponding target sequence. Depending on the nature of the reporter molecule associated with a target polynucleotide, a signal may be instrumentally detected by irradiating a fluorescent label with light and detecting fluorescence in a fluorimeter; by providing for an enzyme system to produce a dye which could be detected using a spectrophotometer; or detection of a dye particle or a coloured colloidal metallic or non metallic particle using a reflectometer; in the case of using a radioactive label or chemiluminescent molecule employing a radiation counter or autoradiography. Accordingly, a detection means may be adapted to detect or scan light associated with the label which light may include fluorescent, luminescent, focussed beam or laser light. In such a case, a charge couple device (CCD) or a photocell can be used to scan for emission of light from a probe:target polynucleotide hybrid from each location in the micro-array and record the data directly in a digital computer. In some cases, electronic detection of the signal may not be necessary. For example, with enzymatically generated colour spots associated with nucleic acid array format, visual examination of the array will allow interpretation of the pattern on the array. In the case of a nucleic acid array, the detection means is suitably interfaced with pattern recognition software to convert the pattern of signals from the array into a plain language genetic profile. In certain embodiments, oligonucleotide probes specific for at least a portion of a prostate cancer marker polynucleotide are in the form of a nucleic acid array and detection of a signal generated from a reporter molecule on the array is performed using a 'chip reader'. A detection system that can be used by a 'chip reader' is described for example by Pirrung et at, (U.S. Patent No. 5,143,854). The chip reader will typically also incorporate some signal processing to determine whether the signal at a particular array position or feature is a true positive or maybe a spurious signal. Exemplary chip readers are described for example by Fodor et al, (U.S. Patent No., 5,925,525). Alternatively, when the array is made using a mixture of individually addressable kinds of labelled microbeads, the reaction may be detected using flow cytometry.

4. Kits

[0169] All the essential materials and reagents required for detecting and quantifying prostate cancer marker polynucleotides of the invention may be assembled together in a kit. The kits may also optionally include appropriate reagents for detecting labels, positive and negative controls, washing solutions, blotting membranes, microtitre plates, dilution buffers and the like. For example, a nucleic acid-based detection kit may include (i) a prostate cancer marker polynucleotide, (ii) a primer or probe that specifically hybridizes to a prostate cancer marker polynucleotide. Also included may be enzymes suitable for amplifying nucleic acids including various polymerase (Reverse Transcriptase, Taq, Sequenase™ DNA ligase etc., depending on the nucleic acid amplification technique employed), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe. The kit can also feature various devices and reagents for performing one of the assays described herein; and/or printed instructions for using the kit to quantify expression of a prostate cancer marker.

5. Methods of treatment or prophylaxis [0170] The present invention also extends to the management of prostate cancer and prostate cancer metastasis, or prevention of further progression of prostate cancer and prostate cancer metastasis, or assessment of the efficacy of therapies in subjects following positive diagnosis for the presence or risk of development of prostate cancer in the subjects. Generally, the management of prostate cancer is highly intensive and can include radiation therapy, surgery, chemotherapy, immunotherapy, hormone ablation therapy and other cancer therapies. It will be understood, however, that the present invention encompasses any agent or process that is useluϊ for treating prostate cancer and prostate cancer metastasis and is not limited to the aforementioned illustrative compounds or strategies.

5.1 Radiotherapy [0171] Radiotherapies include radiation and waves that induce DNA damage for example, γ-irradiation, X rays, UV irradiation, microwaves, electronic emissions, radioisotopes, and the like. Therapy may be achieved by irradiating the localized tumour site with the above described forms of radiations. It is most likely that all of these factors effect a broad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes.

[0172] Dosage ranges for X rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. [0173] Non-limiting examples of radiotherapies include conformal external beam radiotherapy (50-100 Grey given as fractions over 4-8 weeks), either single shot or fractionated, high dose rate brachytherapy, permanent interstitial brachytherapy, systemic radio-isotopes (e.g., Strontium 89). hi some embodiments the radiotherapy may be administered in combination with a radiosensitizing agent. Illustrative examples of radiosensitizing agents include but are not limited to efaproxiral, etanidazole, fluosol, misonidazole, nimorazole, temoporfin and tirapazamine. 5.2 Surgery

[0174] Surgical treatment for removal of a cancerous growth is generally a standard procedure for the treatment of tumours and cancers. This attempts to remove the entire cancerous growth. However, surgery is generally combined with chemotherapy and/or radiotherapy to ensure the destruction of any remaining neoplastic or malignant cells.

5.3 Chemotherapy

[0175] Chemotherapeutic agents may be selected from any one or more of the following categories:

[0176] (i) cytotoxic agents (e.g., antiproliferative/antineoplastic drugs and combinations thereof), as used in medical oncology, such as alkylating agents (for example cis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambμcil, busulphan and nitrosoureas); antimetabolites (for example antifolates such as fluoropyridines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea; anti-tumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like paclitaxel and docetaxel; and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin); [0177] (ii) cytostatic agents such as antioestrogens (for example tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene), oestrogen receptor down regulators (for example fulvestrant), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), UH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5α-reductase such as finasteride;

[0178] (iii) agents that inhibit cancer cell invasion (for example metalloproteinase inhibitors like marimastat and inhibitors of urokinase plasminogen activator receptor function); [0179] (iv) inhibitors of growth factor function, for example such inhibitors include growth factor antibodies, growth factor receptor antibodies (for example the anti-erbb2 antibody trastuzumab [Herceptin™] and the anti-erbbl antibody cetuximab [C225]), farnesyl transferase inhibitors, MEK inhibitors, tyrosine kinase inhibitors and serine/threonine kinase inhibitors, for example other inhibitors of the epidermal growth factor family (for example other EGFR family tyrosine kinase inhibitors such as N-(3-chloro-4-fluorophenyl)-7- methoxy-6-(3-morpholinopropoxy)quinazolin-4- -amine (gefitinib, AZD1839), N-(3- ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) and 6- acrylamido-N-(3 -chloro-4-fluorophenyl)-7-(3 -morpholinopropoxy)quinazoli- n-4-amine (CI 1033)), for example inhibitors of the platelet-derived growth factor family and for example inhibitors of the hepatocyte growth factor family;

[0180] (v) anti-angiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, (for example the ami- vascular endothelial cell growth factor antibody bevacizumab [Avastin™], compounds such as those disclosed in International PatentApplications WO, 97/22596, WO 97/30035, WO 97/32856 and. WO 98/13354) and . compounds that work by other mechanisms (for example linomide, inhibitors of integrin .alpha.v.beta.3 function and angiostatin); [0181] (vi) vascular damaging agents such as Combretastatin A4 and compounds disclosed in International Patent Applications WO 99/02166, WO00/40529, WO 00/41669, WOO 1/92224, WO02/04434 and WO02/08213;

[0182] (vii) antisense therapies, for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense; and [0183] (viii) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCAl or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy.

5.4 Immunotherapy

[0184] Immunotherapy approaches, include for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti-idiotypic antibodies. These approaches generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumour cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumour cell target. Various effector cells include cytotoxic T cells and NK cells.

[0185] In targeting embodiments, the tumour cell generally bears some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many prostate tumour markers exist and any of these may be suitable for targeting in the context of the present invention. Common prostate cancer markers include PSA and other Kallikrein-related proteases, ESMA, PR, AR₅-ER, laminin receptor, erb B andpl55._

5.5 Hormone ablation therapy

[0186] Hormone or androgen ablation therapy refers to techniques for the removal or destruction of sources of male hormones, such as testosterone. These techniques include, for example, 1) surgical removal of the testicles, 2) medications that inhibit testosterone production, or 3) anti-androgenic drugs that block androgen receptors. Chemical agents suitable for use as hormone ablation therapy for prostate cancer include, but are not limited to, non-steroidal anti-androgens such as Nilutamide, Bicalutamide and flutamide; GnRH agonists such as Goserelin acetate, leuprorelin and triptorelin; 5-alpha reductase inhibitors such as finasteride; and cyproterone acetate.

5.6 Anti-resorptive agents

[0187] Anti-resorptive agents or ARAs have several properties including selective uptake at active bone sites, suppression of osteoblast and osteoclast mediated bone resorption, reduction in the number of osteoclasts and long skeletal retention, which have been shown to minimize the destructive consequences of bone metastases and to exert a profound effect on tumour-induced osteolysis and tumour growth in bone. The most common classes of anti- resorptive drugs include oestrogen, selective oestrogen receptor modulators (SERMs), biphosphonates, calcitonin, osteoprotegrin (OPG), cathespin K and statins. Current products include FOSAMAX™ (alendronate) in the U.S., Biphosphonate DIDRONEL™ (etidronate) and ACTONEL™ (risedronate) 5.7 Other Cancer Therapies

[0188] Examples of other cancer therapies include phototherapy, cryotherapy, toxin therapy or pro-apoptosis therapy . One of skill in the art would know that this list is not exhaustive of the types of treatment modalities available for cancer and other hyperplastic lesions.

[0189] Typically, the therapeutic agents will be administered in pharmaceutical compositions together with a pharmaceutically acceptable carrier and in an effective amount to achieve their intended purpose. The dose of active compounds administered to a subject should be sufficient to achieve a beneficial response in the subject over time such as a reduction in, or relief from, the symptoms of prostate cancer. The quantity of the pharmaceutically active compounds(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general heakh-condition-thereofτ In this-regard,- precise amounts of the active compound(s) for administration will depend on the judgement of the practitioner. In determining the effective amount of the active compound(s) to be administered in the treatment of prostate cancer, the medical practitioner or veterinarian may evaluate severity of any symptom associated with the presence of prostate cancer. In any event, those of skill in the art may readily determine suitable dosages of the therapeutic agents and suitable treatment regimens without undue experimentation.

[0190] Exemplary subjects for treatment with the methods of the present invention are vertebrates, especially mammals. In certain embodiments, the subject is selected from the group consisting of humans, sheep, cattle, horses, bovine, pigs, dogs and cats. In specific embodiments, the subject is a human.

[0191] In some embodiments, a patient's PCA3 expression profile is assessed in combination with at least one ancillary cancer factor to correlate disease outcome for the patient. Non-limiting examples of ancillary cancer factors include the subject's pre-treatment PSA; the subject's post-treatment PSA; primary Gleason grade in a biopsy specimen obtained from the subject; secondary Gleason grade hi a biopsy specimen obtained from the subject; Gleason sum in a biopsy specimen obtained from the subject; pre-radical primary therapy of the subject; total length of cancer in biopsy cores obtained from the subject; number of positive biopsy cores obtained from the subject; percent of tumour biopsy in a multiple core biopsy set obtained from the subject; primary Gleason grade in a pathological specimen obtained from the subject; secondary Gleason grade in a pathological specimen obtained from the subject; Gleason sum in a pathological specimen obtained from the subject; the subject's pre-operative TGF-ssl level; the subject's prostatic capsular invasion level (PCI); the subject's surgical margin status; the subject's seminal vesicle involvement; the subject's lymph node status; the subject's pre-operative IL6sR level; the sensitivity of the subject's cancer to hormone therapy; the resistance of the subject's cancer to hormone therapy; the subject's prior therapy and/or clinical stage. Other non-limiting ancillary cancer factors may be selected from expression profiles of hormone-related cancer maker genes, illustrative examples of which include PCA3, Claudin 4, Hepsin, PSMA, SPINKl, GOLPH2, TMPRSS2.ERG, GalNAc-T3, HER2 /neu/ERbB2, Cathepsin D, BRCAl, BRCA2, ER, PR, AR, MUCl, EGFR, mutant p53, cyclin D, PCNA, Ki67, uPA and PAI. [0192] Disease outcome may be defined according to at least one of: probability of disease recurrence or metastasis at one or a plurality of time points between one and twenty years .e.g., five, ten£>r_fϊfteen years-following a primary treatment; estimated.disease or . metastasis free survival; organ confined disease; extracapsular extension; seminal vesical involvement and lymph node status in the patient following radical prostatectomy. [0193] The functional correlation between disease outcome, PCAS expression profiles and the one or more ancillary cancer factors may be generated by any means known in the art. For example, the functional correlation may be generated by a means selected from the group consisting of a neural network, Cox proportional hazards regression model and support vector machine. In some embodiments, the correlation is generated by computer and/or software means.

[0194] Thus, in operation, when a PCA3 expression profile, either alone or in combination with one or more ancillary cancer factors, correlates with a negative disease outcome for a patient, the patient may be administered additional or more aggressive cancer therapies, non-limiting examples of which include radiotherapy, surgery, chemotherapy, hormone ablation therapy, pro-apoptosis therapy and immunotherapy, as discussed for example above.

[0195] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non- limiting examples. EXAMPLES

EXAMPLE l

IDENTIFICATION OF TRANSCRIPTION START SITES PCA3-3, PCA3-4, PCA3-5 AND PCA3-6

AND NOVEL PCA3 TRANSCRIPTS Materials and Methods

[0196] High quality total RNA was extracted from 1 μg of prostate tissue using Trizol (Invitrogen) following the manufactures instructions. The extracted RNA was reverse transcribed using 1 μl of SuperscriptIII Reverse Transcriptase (10000U) (Invitrogen), where each reaction was primed with 1 μl of 12 μM 5'-CDS primer A (5'-(T)₂₅VN-3') (Clontech) and 1 μl of 12 μM SMART II A oligo (5'-AAGCAGTGGTATCAA CGCAGAGT

ACGCGGG-3'^"; SEQTD NO: 63) (Clontech). After ^"aimealing^'όf the^'hexanucϊeotidVs for 10^{" " ~} minutes at 70° C, cDNA synthesis was performed at 42° C for 90 minutes followed by an enzyme inactivation step of 72⁰C for 7 minutes, after adding 100 μl of Tricine-EDTA buffer. The 5' RACE clones were amplified with 5' RACE PC A3 primers as follows: [0197] (A). 5'-GCAGGTGGCCACTCCCA TCATG CAAG - 3' (SEQ ID NO: 59; complementary to nucleotides 1096-1121 of SEQ ID NO: 15);

[0198] (B). 5 '- TCACACTATGTTGAC ACGTTG CTA -3 ' (SEQ ID NO: 60; complementary to nucleotides 991-1014 of SEQ ID NO: 15);

[0199] (C). 5'- TGAGATCCACAGGTCA ATTCCTA - 3'(SEQ ID NO: 61; complementary to nucleotides 707-729 of SEQ ID NO: 15); and

[0200] (D). 5'- GCACTTCCTGTCACTGCCA TACCA -3' (SEQ ID NO: 62; complementary to nucleotides 427-450 of SEQ ID NO: 15).

[0201] In addition, 1OX Universal Primer A mix (Clontech) was added following the manufacturer's instructions (Clontech). [0202] The following PCR program was applied: 5 cycles of 94° C for 30 seconds and 72° C for 2 minutes; 5 cycles of 94° C for 30 seconds, 70° C for 30 seconds and 72° C for 2 minutes; and 30 cycles of 94° C for 30 seconds, 68° C for 30 seconds and 72° C for 2 minutes. The 5' RACE PCR products that were generated were excised and cloned into a pGEMT vector. Positive clones that were generated, were then sequenced using sequencing techniques known in the art. [0203] Sequencing results obtained were blasted against the NCBI human genome database (http://wmv.ncbi.nlm.nih.gov/blast/Blast.cgO. Sequencing results from L2 and L3 clones as illustrated by Figure 3 (gel A) identified PCA3 transcription start sites PC A3 -6 and PCA3-5. Ll generated a non-specific product. To confirm the results, 5'RACE from further prostate cancer and metastatic tissues was carried out. Sequencing results from L4, L5, L6 and L7 clones as illustrated by Figure 3 (gel B) identified PC A3 transcription start site PCA3- 6. This result is consistent with the sequencing result of L2. L8 and L9 clones generated a non-specific product. Sequencing results of LlO identified transcription start site PCA3-4. To identify further PC A3 transcription start sites, three different 5¹RACE primers (A, B and C) were designed upstream of primer A. Figure 3 (gel C) illustrates that primers B and C did not identify any PCA3 positive clones, whereas Primer D sequencing results identified PCA3 transcription start site PCA3-3. Positive clones were only generated in metastatic and prostate cancer tissue samples but not in BPH samples.

[0204] Using 5'RACE primer (A) as described above, 3 transcription start sites (PCA3-4, PCA3-5 and PCA3-6) were identified (see Figure 3). No start site was identified when primers (B) and (C) were used. With primer (D), transcription start site PCA3-3 was identified. A diagrammatic representation of the novel PC A3 transcriptional units is presented in Figure 2.

[0205] Prostate tissue samples were taken from a number of subjects wherein their status was either cancerous, benign or metastatic and their Gleason score was measured as shown in Table 1 below.

Table 1

EXAMPLE 2 DETECTION OF PROSTATE CANCER IN A BIOLOGICAL TISSUE SAMPLE FROM A SUBJECT

Tissue Collection

[0206] All tissue specimens were collected from consenting patients at Royal Brisbane and Woman's Hospital, Queensland, as approved by the Institutional Ethics Committee. Primary prostate cancer tissue specimens were obtained from patients undergoing radical prostatectomy with secondary tumours harvested from lymph node metastases in patients with hormone refractory prostate cancer. Benign prostatic hyperplasia (BPH) tissue specimens were obtained from men who underwent either transurethral resection of the prostate (TURP) or an open enucleative prostatectomy. Tissue fragments were frozen immediately using liquid nitrogen and transported on dry ice for storage at -70° with closely adjacent tissue specimens placed in Ornithine Carbamyl Transferase (OCT) and snap frozen or formalin fixed and paraffin-embedded. Tissues prepared for histology were examined to confirm the diagnosis of BPH or prostate cancer and to determine the proportion of epithelial cells to stromal cells.

RNA isolation and cDNA synthesis

[0207] Total RNA was extracted from prostate tissues using Trizol (Invitrogen) following manufacture protocol. Subsequent DNase treatment was performed with DNase I (NEB Biolabs: Cat No. M0303S), ethanol precipitated, resuspended in DEPC-treated water and quality controlled via spectrophotometry and gel electrophoresis. All RNA was confirmed to be of good quality and thus suitable for subsequent experiments if the A260/280 ratio was >1.7 and little RNA degradation was evident by gel electrophoresis, lμg of total RNA extracted was reverse transcribed using 250ng of random hexamers (Promega) in a standard 20 μl reaction including 4μl of first strand buffer (Invitrogen), 2 μl of 0.1 M DDT (Invitrogen), 1 μl of 1OmM dNTP (Promega), 1 μl RNase inhibitor^" (2500U) (Promega) and 1 μl of reverse transcriptase (10,000U) (Invitrogen). After annealing of the hexanucleotides for 10 minutes at 72⁰C, cDNA synthesis was performed for 42⁰C for 90 minutes followed by an enzyme inactivation step at 7O⁰C for 15 minutes. All cDNA products were diluted in a ratio of 1 : 10 and stored at -2O⁰C before use.

Non-Quantitative PCR measurements

[0208] Non-quantitative RTPCR was carried out on a PCR thermocycler (MJ research) with gene specific primers (see below). Each reaction contained 5 μl of the diluted cDNA template, 2.5 μl of 1OX PCR buffer, 0.2 μl of 25 mM dNTPs, 1 μl of each of the forward and reverse primer stocks (1OmM), 1.5 μl of 25 mM MgCl₂ and 0.25 μl of AmpliTaq Gold polymerase (Applied Biosystems). The following PCR conditions were applied: initial denaturation of 94°C for 10 minutes followed by 40 cycles of 94C° for 30 seconds, 58°C for 30 seconds and 72°C for 40 seconds and a final extension of 72°C for 15 minutes. [0209] Gene specific primers

[0210] (a) PCA3-4F, 5'-TATTCTGAAGTCAGAGTGTTCCAG-S' (SEQ ID NO: 46; corresponding to nucleotides 1127 to 1150 of SEQ ID NO: 15);

[0211] (b) Ex2cR, S'-ACTCAGAAAGTGCCGTCGAT-S' (SEQ ID NO: 47; complementary to nucleotides 1558 to 1577 of SEQ ID NO: 15); [0212] (c) Ex2aR, 5'-GTACCTGCCTTCATGTCACATTG-S' (SEQ ID NO: 48; corresponding to nucleotides 1325 to 1347 of SEQ ID NO:15). [0213] (d) Primer exon 1 Fwd, 5'- AGAAATAGCAAGTGCCGAG AA -3' (SEQ ID NO: 49; corresponding to nucleotides 1156 to 1176 of SEQ ID NO: 15);

[0214] (e) Primer exon 2c Rvs, 5'- ACTCAGAAAGTGCCGTCGAT -3' (SEQ ID NO: 50; complementary to nucleotides 1372 to 1390 of SEQ ID NO: 15); [0215] (f) Primer 1 Fwd , 5' - TATTCTGAAGTCAGAGTGTTCCAG - 3' (SEQ

ID NO: 51; corresponding to nucleotides 1127 to 1150 of SEQ ID NO: 15); and

[0216] (g) Primer exon 1/3 Rvs, 5' -CTTATTTCTCACCTCTGTATCATCAGG - 3' (SEQ ID NO: 52; complementary to nucleotides 1256 to 1282 of SEQ ID NO: 15).

[0217] Housekeeping/Reference genes: [0218] (i) B2MF, 5'- GTCTTTCTATC TCTTGTACTACACTGAA -3' (SEQ ID

NO: 55), -

[0219] (ii) B2MR, 5' - AACTATCTTGGGCTGTGACAAAG-3' (SEQ ID NO: 56),

[0220] (iii) PSAF, 5 ' - GCATCAGGAACAAAAGCGTG-3 ' (SEQ ID NO: 57); [0221] (iv) PSAR, 5 ' - CCTGAGGAATCGATTCTTCA-3 ' (SEQ ID NO: 58).

[0222] Non-quantitative PCR was carried out using a PCR machine (MJ research) with gene specific primers. The gene specific primers consisted of primer pairs (a)/(b) and (a)/(c) as described above, which amplified at least a portion of the PC A3 transcript with exon 2a. Each reaction contained 5 μl of the diluted cDNA template, 2.5 μl of 1OX PCR buffer, 0.2μl of 25mM dNTP, 1 μl of each 1OmM forward and reverse primers, 1.5 μl of 25 mM

MgCl₂ and 0.25 μl of AmpliTaq Gold polymerase (Applied Biosystems). The following PCR conditions were applied: initial denaturation of 94° C for 10 minutes followed by 40 cycles of 94° C for 30 seconds, 58° C for 30 seconds and 72° C for 40 seconds and a final extension of 72⁰ C for 15 minutes. [0223] Figure 4 illustrates that non-quantitative PCR can detect prostate cancer and/or prostate cancer metastasis hi tissue from subjects with prostate cancer and/or prostate cancer metastasis using the methods and agents of the invention. Lanes 1 to 3 of gel (I) represent amplified transcripts that contain exons 2a and 2b, amplified using primer pairs (a) and (b) detailed above. Lanes 1 to 3 of gel (II) represent amplified transcripts that contain exon 2a, amplified using primer pairs (a) and (c) detailed above. The transcripts were differentially expressed in tissue samples with BPH compared to tissue samples comprising prostate cancer or prostate cancer metastasis.

[0224] Figure 5 illustrates that non-quantitative PCR can detect tumours in prostate tissue from subjects with prostate cancer using the methods and agents of the invention. Lanes 1 and 2 represent amplified transcripts that contain exon 2c, amplified using primer pairs (d)/(e) and (f)/(g) respectively, detailed above. The transcripts were differentially expressed in tissue samples with BPH compared to tissue samples comprising prostate cancer and/or metastasis. The art known PCA3 biomarker (Lane 3) amplified by the art known primers (exon 1 Fwd; SEQ ID NO: 53 and exon 4 Rvs; SEQ ID NO: 54) also show no discrimination between BPH and prostate cancer (tumours and metastasis) in prostate cancer.

Quantitative PCR measurements

[0225] The cDNA-specific real-time quantitative PCR (QPCR) assay was carried out on the Corbett Rotor-Gene 3000 (Corbett Research, Australia) with gene specific primers as illustrated above for the non-quantitative PCR (and QIAGEN SYBR-GREEN qPCR Mastermix (QIAGEN, Germany). Each reaction contained 7.5 μl of qPCR mastermix, 5pmol of each forward and reverse primer and 5 μl of the diluted cDNA template. The following cycling conditions were applied: 95° C for 15 minutes, followed by 40 cycles of 95° C for 20 seconds, 58-59° C for 20 seconds and 72° C for 20 seconds. Data for each cycle was acquired at the 72° C for 20 second step. Each reaction was carried out in triplicates (subject samples) and triplicate (calibrator).

[0226] A method for calculating relative gene expression, previously described by Pfaffl, M. W (2001, Nucleic Acid Res, 29(9): e45), was applied in the analysis of the realtime PCR results. The Pfaffl, 2001 equation is as follows:

[0227] Expression (R) = EGOIΔCt (Calibrator-sample)/ERefΔCt (Calibrator- sample)

[0228] ΔCt (calibrator-sample) in this equation refers to the Ct deviation between the target gene transcription of the sample (unknown) reaction and the calibrator (uniform template quantity to standardize all the runs) reaction (Pfaffl, 2001 (supra)). This method took into account the difference in amplification and reaction efficiency (E) of the gene of interest (GOI) and the endogenous reference gene (i.e., B2M). This method also eliminates the use of a standard curve in every run by assuming the reaction efficiency between different runs was consistent and normalized by the calibrator used. [0229] The results illustrated by Figure 5 indicate that QPCR is more sensitive than non-quantitative PCR, but both techniques are capable of distinguishing between prostate cancer and BPH using the methods and agents of the invention.

EXAMPLE 3 DETECTION OF PROSTATE CANCER IN A BIOLOGICAL TISSUE SAMPLE FROM A SUBJECT

Tissue Collection

[0230] All tissue specimens were collected from consenting patients at Royal Brisbane and Woman's Hospital, Queensland, as approved by the Institutional Ethics Committee. Primary prostate cancer tissue specimens were obtained from patients undergoing radical prostatectomy with secondary tumours harvested from lymph node metastases in patients with hormone refractory prostate cancer. Benign prostatic hyperplasia (BPH) tissue specimens were obtained from men who underwent either transurethral resection of the prostate (TURP) or an open enucleative prostatectomy. Tissue fragments were frozen immediately using liquid nitrogen and transported on dry ice for storage at -70° with closely adjacent tissue specimens placed in OCT and snap frozen or formalin fixed and paraffin- embedded. Tissues prepared for histology were examined to confirm the diagnosis of BPH or prostate cancer and to determine the proportion of epithelial cells to stromal cells.

RNA isolation and cDNA synthesis

[0231] Total RNA was extracted from prostate tissues using Trizol (Invitrogen) following manufacture protocol. Subsequent DNase treatment was performed with DNase I (NEB Biolabs: Cat No. M0303S), ethanol precipitated, resuspended in DEPC-treated water and quality controlled via spectrophotometry and gel electrophoresis. AU RNA was confirmed to be of good quality and thus suitable for subsequent experiments if the A260/280 ratio was >1.7 and little RNA degradation was evident by gel electrophoresis, lμg of total RNA extracted was reverse transcribed using 250ng of random hexamers (Promega) in a standard 20 μl reaction including 4μl of first strand buffer (Invitrogen), 2 μl of 0.1 M DDT (Invitrogen), 1 μl of 1OmM dNTP (Promega), 1 μl RNase inhibitor (2500U) (Promega) and 1 μl of reverse transcriptase (10,000U) (Invitrogen). After annealing of the hexanucleotides for 10 minutes at 72oC, cDNA synthesis was performed for 42°C for 90 minutes followed by an enzyme inactivation step at 70°C for 15 minutes. All cDNA products were diluted in a ratio of 1 : 10 and stored at -20°C before use. Non-Quantitative PCR measurements

[0232] Non-quantitative RT-PCR was carried out on a PCR thermocycler (MJ research) with gene specific primers (see below). Each reaction contained 5 μl of the diluted cDNA template, 2.5 μl of 1OX PCR buffer, 0.2 μl of 25 mM dNTPs, 1 μl of each of the forward and reverse primer stocks (1OmM), 1.5 μl of 25 mM MgCl₂ and 0.25 μl of AmpliTaq Gold polymerase (Applied Biosystems). The following PCR conditions were applied: initial denaturation of 94°C for 10 minutes followed by 35 cycles of 94°C for 30 seconds, 64°C for 30 seconds and 72°C for 40 seconds and a final extension of 72°C for 15 minutes.

[0233] (a) Ex2AF, 5 ' - GAGTCCCTGCTC AAGGAGAC AGAC AC AAAC -3 ' (SEQ ID NO: 64; corresponding to nucleotides 1290 to 1319 of SEQ ID NO: 15);

[0234] (b) Ex2BF, 5 ' - GTC AC AAACTTCTC AGCTTAAGC AATCTGC -3 ' (SEQ ID NO: 66; corresponding to nucleotides 1394 to 1423 of SEQ ID NO: 15);

[0235] (c) Ex3R, 5' - GTGTGGCCTCAGATGGTAAAGTCAGCAGC -3'(SEQ ID NO: 65; complementary to nucleotides 1638 to 1666 of SEQ ID NO: 15); [0236] Figure 6 illustrates that non-quantitative PCR can detect prostate cancer and metastasis in tissue from subjects with prostate cancer using the methods and agents of the invention. BPH, PCA and METS sample lanes of gel (i) represent amplified transcripts that contain exon 2a, amplified using primer pairs (a) and (c) detailed above. BPH, PCA and METS sample lanes of gel (i) represent amplified transcripts that contain exon 2b, amplified using primer pairs (b) and (c) detailed above. Both transcripts were differentially expressed in tissue samples with BPH compared to tissue samples comprising prostate cancer or metastases. From the results, the novel exon2a and exon 2b appear to be useful markers for prostate cancer diagnosis.

EXAMPLE 4

DETECTION OF PROSTATE CANCER IN A BIOLOGICAL PROSTATIC FLUID SAMPLE FROM A

SUBJECT

Prostatic Fluid Collection

[0237] All prostatic fluid specimens were collected from consenting patients at Royal Brisbane and Woman's Hospital, Queensland, as approved by the Institutional Ethics Committee. Following informed consent, prostatic fluid will be obtained in the forms of post- DRE urine, seminal fluid, and post-ejaculate urine. The initial stage of isolating mononuclear cells from ejaculate samples collected in 20 ml Hanks buffer (Invitrogen, Victoria, Australia); the sample is carefully layered over isotonic Percoll (Amersham Biosciences, NSW) and centrifuged permitting the mononuclear cells to be collected from the Percoll/Sample interface which are then resuspended and washed in PBS. Trizol were added to the mononuclear cells for RNA extraction.

RNA isolation and cDNA synthesis

[0238] Total RNA was extracted from the mononuclear cells using Trizol (Invitrogen) following manufacture protocol. Subsequent DNase treatment was performed with DNase I (NEB Biolabs: Cat No. M0303S), ethanol precipitated, resuspended in DEPC- treated water and quality controlled via spectrophotometry and gel electrophoresis. AU RNA was confirmed to be of good quality and thus suitable for subsequent experiments if the A260/280 ratio was >1.7 and little RNA degradation was evident by gel electrophoresis, lμg of total RNA extracted was reverse transcribed using 250ng of random hexamers (Promega) in a standard 20 μl reaction including 4μl of first strand buffer (Invitrogen), 2 μl of 0.1M DDT (Invitrogen), 1 μl of 1OmM dNTP (Promega), 1 μl RNase inhibitor (2500U) (Promega) and 1 μl of reverse transcriptase (10,000U) (Invitrogen). After annealing of the hexanucleotides for 10 minutes at 72°C, cDNA synthesis was performed for 42°C for 90 minutes followed by an enzyme inactivation step at 70°C for 15 minutes. All cDNA products were diluted in a ratio of 1 :10 and stored at -20°C before use. Quantitative PCR measurements

[0239] The cDNA-specific real-time quantitative PCR assay was carried out on the Corbett Rotor-Gene 3000 (Corbett Research, Australia) with gene specific primers (Reference gene PSA and test gene PCA3 isoform primers) and QIAGEN SYBR-GREEN qPCR Mastermix (QLAGEN, Germany).Each reaction contained 7.5 μl of qPCR mastermix, 5pmol of each forward and reverse primer and 5μl of the diluted cDNA template. The following cycling conditions were applied: 95⁰C for 15 minutes, followed by 45 cycles of 95⁰C for 20 seconds, 58-59⁰C for 20 seconds and 72⁰C for 20 seconds. Data for each cycle was acquired at the 72⁰C for 20 second step. Each reaction was carried out in triplicates (patient samples) and triplicate (calibrator). A method for calculating relative gene expression, previously described by Pfaffl (2001, supra), was applied in the analysis of the real-time PCR results.

[0240] The ROC curve was used to determine the sensitivity and specificity of PCA3 isoform in detecting prostate cancer (Figure 7). The results show that each of the three PCA3 isoforms tested is useful for discriminating prostate cancer and prostate cancer metastasis from BPH.

[0241] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety. [0242] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

[0243] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for diagnosing the presence or risk of development of prostate cancer in a subject, the method comprising detecting in the subject expression of a PCA3 nucleic acid sequence comprising exon 2a.

2. The method according to claim 1, wherein the exon 2a comprises the sequence set forth in SEQ ID NO: 6, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 6, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 6 under at least high stringency conditions.

3. The method according to claim 1, wherein the expressed nucleic acid sequence further comprises an exon 1 sequence.

- -

4. The method according to claim 3, wherein the exon 1 sequence is selected from the group consisting of: an exon Ia sequence, an exon Ib sequence, an exon Ic sequence, an exon Id sequence and an exon Ie sequence.

5. The method according to claim 4, wherein the exon Ia sequence comprises the sequence set forth in SEQ ID NO: 1 or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 1, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 1 under at least high stringency conditions.

6. The method according to claim 4, wherein the exon Ib sequence comprises the sequence set forth in SEQ ID NO: 2 or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 2, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 2 under at least high stringency conditions.

7. The method according to claim 4, wherein the exon Ic sequence comprises the sequence set forth in SEQ ED NO: 3 or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 3, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 3 under at least high stringency conditions.

8. The method according to claim 4, wherein the exon Id sequence comprises the sequence set forth in SEQ ID NO: 4 or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 4, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 4 under at least high stringency conditions.

9. The method according to claim 4, wherein the exon Ie sequence comprises the sequence set forth in SEQ ID NO: 5 or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 5, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 5 under at least high stringency conditions.

10. The method according to claim 1, wherein the expressed nucleic acid sequence further comprises an exon 2b sequence.

11. The method according to claim 10, wherein the exon 2b sequence comprises the sequence set forth in SEQ ID NO: 7.

12. The method according to claim 1, wherein the expressed nucleic acid sequence further comprises an exon 2c sequence.

13. The method according to claim 12, wherein the exon 2c sequence comprises the sequence set forth in SEQ ID NO: 8.

14. The method according to claim 1, wherein the expressed nucleic acid sequence further comprises an exon 3 sequence.

15. The method according to claim 14, wherein the exon 3 sequence comprises the sequence set forth in SEQ ID NO: 9.

16. The method according to claim 1, wherein the expressed nucleic acid sequence further comprises an exon 4 sequence.

17. The method according to claim 16, wherein the exon 4 sequence comprises the sequence set forth in SEQ ID NO: 10.

18. The method according to any one of claims 1 to 17, wherein the expressed nucleic acid sequence comprises an exon 1 sequence, an exon 2b sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence.

19. The method according to claim 18, wherein the expressed nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NO: 11-15.

20. The method according to any one of claims 1 to 17, wherein the expressed nucleic acid sequence comprises an exon 1 sequence, an exon 2b sequence, an exon 3 sequence and an exon 4 sequence.

21. The method according to claim 20, wherein the expressed nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NO: 16-20.

22. The method according to any one of claims 1 to 17, wherein the expressed nucleic acid sequence comprises an exon 1 sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence.

23. The method according to claim 22, wherein the expressed nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NO: 21-25.

24. The method according to any one of claims 1 to 17, wherein the expressed nucleic acid sequence comprises an exon 1 sequence, an exon 3 sequence and an exon 4 sequence.

25. The method according to claim 24, wherein the expressed nucleic acid sequence comprises the sequence set forth hi any one of SEQ ID NO: 31-35

26. A method for diagnosing the presence or risk of development of prostate cancer in a subject, the method comprising detecting in the subject expression of a PCA3 nucleic acid sequence comprising exon 2b.

27. The method according to claim 26, wherein the exon 2b comprises the sequence set forth in SEQ ID NO: 7, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 7, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 7 under at least high stringency conditions.

28. The method according to claim 26, wherein the expressed nucleic acid sequence further comprises an exon 1 sequence.

29. The method according to claim 28, wherein the exon 1 sequence is selected from the group consisting of: an exon Ia sequence, an exon Ib sequence, an exon Ic sequence, an exon Id sequence and an exon Ie sequence.

30. The method according to claim 29, wherein the exon Ia sequence comprises the sequence set forth in SEQ ID NO: 1, or a sequence that displays at least

90% sequence identity to the sequence set forth in SEQ ID NO: 1, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 1 under at least high stringency conditions.

31. The method according to claim 29, wherein the exon Ib sequence comprises the sequence set forth in SEQ ID NO: 2, or a sequence that displays at least

90% sequence identity to the sequence set forth in SEQ ID NO: 2, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 2 under at least high stringency conditions.

32. The method according to claim 29, wherein the exon Ic sequence comprises the sequence set forth in SEQ ID NO: 3, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 3, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 3 under at least high stringency conditions.

33. The method according to claim 29, wherein the exon Id sequence comprises the sequence set forth in SEQ ID NO: 4, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 4, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 4 under at least high stringency conditions.

34. The method according to claim 29, wherein the exon Ie sequence comprises the sequence set forth in SEQ ID NO: 5, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 5, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 5 under at least high stringency conditions.

35. The method according to claim 26, wherein the expressed nucleic acid sequence further comprises an exon 2a sequence.

36. The method according to claim 35, wherein the exon 2a sequence comprises the sequence set forth in SEQ ID NO: 6.

37. The method according to claim 26, wherein the expressed nucleic acid sequence further comprises an exon 2c sequence.

38. The method according to claim 37, wherein the exon 2c sequence comprises the sequence set forth in SEQ ID NO: 8.

39. The method according to claim 26, wherein the expressed nucleic acid sequence further comprises an exon 3 sequence.

40. The method according to claim 39, wherein the exon 3 sequence comprises the sequence set forth in SEQ ID NO: 9.

41. The method according to claim 26, wherein the expressed nucleic acid sequence further comprises an exon 4 sequence.

42. The method according to claim 41, wherein the exon 4 sequence comprises the sequence set forth in SEQ ID NO: 10.

43. The method according to any one of claims 26 to 42, wherein the expressed nucleic acid sequence comprises an exon 1 sequence, an exon 2a sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence.

44. The method according to claim 43, wherein the expressed nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NO: 11-15.

45. The method according to any one of claims 26 to 42, wherein the expressed nucleic acid sequence comprises an exon 1 sequence, an exon 2a sequence, an exon 3 sequence and an exon 4 sequence.

46. The method according to claim 45, wherein the expressed nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NO: 16-20.

47. The method according to any one of claims 26 to 42, wherein the expressed nucleic acid sequence comprises an exon 1 sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence.

48. The method according to claim 47, wherein the expressed nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NO: 26-30.

49. The method according to any one of claims 26 to 42, wherein the expressed nucleic acid sequence comprises an exon 1 sequence, an exon 3 sequence and an exon 4 sequence.

50. The method according to claim 49, wherein the expressed nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NO: 36-40.

51. A method for diagnosing the presence or risk of development of prostate cancer in a subject, the method comprising detecting in the subject expression of a PCA3 nucleic acid sequence comprising exon 2c.

52. The method according to claim 26, wherein the exon 2c comprises the sequence set forth in SEQ ID NO: 8, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 8, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 8 under at least high stringency conditions.

53. The method according to claim 51, wherein the expressed nucleic acid sequence further comprises an exon 1 sequence.

54. The method according to claim 53, wherein the exon 1 sequence is selected from the group consisting of: an exon Ia sequence, an exon Ib sequence, an exon Ic sequence, an exon Id sequence and an exon Ie sequence.

55. The method according to claim 54, wherein the exon Ia sequence comprises the sequence set forth in SEQ ID NO: 1, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 1, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 1 under at least high stringency conditions.

56. The method according to claim 54, wherein the exon Ib sequence comprises the sequence set forth in SEQ ID NO: 2, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 2, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 2 under at least high stringency conditions.

57. The method according to claim 54, wherein the exon Ic sequence comprises the sequence set forth in SEQ ID NO: 3, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 3, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 3 under at least high stringency conditions.

58. The method according to claim 54, wherein the exon Id sequence comprises the sequence set forth in SEQ ID NO: 4, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 4, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 4 under at least high stringency conditions.

59. The method according to claim 54, wherein the exon Ie sequence comprises the sequence set forth in SEQ ID NO: 5, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 5, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 5 under at least high stringency conditions.

60. The method according to claim 51, wherein the expressed nucleic acid sequence further comprises an exon 2a sequence.

61. The method according to claim 60, wherein the exon 2a sequence comprises the sequence set forth in SEQ ID NO: 6.

62. The method according to claim 51, wherein the expressed nucleic acid sequence further comprises an exon 2b sequence.

63. The method according to claim 62, wherein the exon 2c sequence comprises the sequence set forth in SEQ ID NO: 7.

64. The method according to claim 51, wherein the expressed nucleic acid sequence further comprises an exon 3 sequence.

65. The method according to claim 64, wherein the exon 3 sequence comprises the sequence set forth in SEQ ID NO: 9.

66. The method according to claim 51, wherein the expressed nucleic acid sequence further comprises an exon 4 sequence.

67. The method according to claim 66, wherein the exon 4 sequence comprises the sequence set forth in SEQ ID NO: 10.

68. The method according to any one of claims 51 to 67, wherein the expressed nucleic acid sequence comprises an exon 1 sequence, an exon 2a sequence, an exon 2b sequence, an exon 3 sequence and an exon 4 sequence.

69. The method according to claim 68, wherein the expressed nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NO: 11-15.

70. The method according to any one of claims 51 to 67, wherein the expressed nucleic acid sequence comprises an exon 1 sequence, an exon 2a sequence, an exon 3 sequence and an exon 4 sequence.

71. The method according to claim 70, wherein the expressed nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NO: 21-25.

72. The method according to any one of claims 51 to 67, wherein the expressed nucleic acid sequence comprises an exon 1 sequence, an exon 2b sequence, an exon 3 sequence and an exon 4 sequence.

73. The method according to claim 72, wherein the expressed nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NO: 26-30.

74. The method according to any one of claims 51 to 67, wherein the expressed nucleic acid sequence comprises an exon 1 sequence, an exon 3 sequence and an exon 4 sequence.

75. The method according to claim 74, wherein the expressed nucleic acid sequence comprises the sequence set forth in any one of SEQ ID NO: 41-45..

76. The method according to any one of claims 1 to 75, comprising: (1) providing a biological sample from the subject; (2) measuring in the biological sample the level of a prostate cancer marker polynucleotide; and (3) comparing the measured level of the PCA3 nucleic acid sequence to the level of a corresponding PCA3 nucleic acid sequence in a reference sample obtained from one or more normal subjects or from one or more subjects lacking prostate cancer, wherein a difference in the level of the PCA3 nucleic acid sequence in the biological sample as compared to the level of the corresponding PCA3 nucleic acid sequence in the reference sample is indicative of the presence or risk of development of prostate cancer in the subject.

77. The method according to any one of claims 1 to 75, comprising: (1) providing a biological sample from the subject; (2) measuring in the biological sample the level of a prostate cancer marker polynucleotide; and (3) comparing the measured level of the PCA3 nucleic acid sequence to the level of a corresponding PCA3 nucleic acid sequence in a reference sample obtained from one or more subjects with BPH, wherein a difference in the level of the PC A3 nucleic acid sequence in the biological sample as compared to the level of the corresponding PCA3 nucleic acid sequence in the reference sample is indicative of the presence or risk of developing prostate cancer in the subject.

78. The method according to claim 76 or claim 77, further comprising: diagnosing the absence of prostate cancer when the measured level of the PCA3 nucleic acid sequence is the same as or similar to the measured level of the corresponding PCA3 nucleic acid sequence.

79. The method according to any one of claims 1 to 78, further comprising: detecting expression of at least one other prostate cancer marker polynucleotide.

80. The method according to claim 79, wherein the other prostate cancer marker polynucleotide is selected from PCA3 (Prostate Cancer Antigen 3), Claudin 4, Hepsin, PSMA (Prostate Specific Membrane Antigen), SPINKl (Serine Peptidase INhibitor, Kazal type 1), GOLPH2 (GOLgi PHosphoprotein 2), KLK2 (KaLHKrein 2), KLK4 (KaLHKrein 4), KLKIl (KaLHKrein 11), KLK14 (KaLHKrein 14), KLK15 (KaLHKrein 15), PBOVl (Prostate and Breast cancer OVerexpressed 1) / UROC28 , BCL2 (B-cell CLL/lymphoma 2), TMPRSS2.ERG, GalNAc-T3 (UDP-N-acetyl-alpha-D- GALactosamine .polypeptide N-ACetylgalactosaminylTransferase 3), MUCl (Mucin 1), EGFR, mutant p53, cyclin D, PCNA, Ki67, uPA, PAI (Plasminogen), HER2 (Human Epidermal growth factor Receptor 2) / neu / ERbB2, Cathepsin D, AR (Androgen Receptor), MUCl (MUCinl), EGFR (Epidermal Growth Factor Receptor), mutant p53, cyclin D, PCNA (Proliferating Cell Nuclear Antigen), Ki67, uPA (urokinase type Plaminogen Activator) and PAI (Plaminogen Activator Inhibitor).

81. The method according to any one of claims 1 to 78, wherein the cancer is primary prostate cancer.

82. The method according to any one of claims 1 to 78, wherein the cancer is secondary prostate cancer.

83. An isolated polynucleotide that consists essentially of at least a portion of a nucleic acid sequence selected from SEQ ID NO: 6, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 6, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 6 under at least high stringency conditions.

84. The polynucleotide according to claim 83, further comprising an exon Ia sequence.

85. The polynucleotide according to claim 83, further comprising an exon Ib sequence.

86. The polynucleotide according to claim 83, further comprising an exon Ic sequence.

87. The polynucleotide according to claim 83, further comprising an exon Id sequence.

88. The polynucleotide according to claim 83, further comprising an exon Ie sequence.

89. The polynucleotide according to claim 83, further comprising an exon 2b sequence.

90. The polynucleotide according to claim 83, further comprising an exon 2c sequence.

91. The polynucleotide according to claim 83, further comprising an exon 3 sequence.

92. The polynucleotide according to claim 83, further comprising an exon 4 sequence.

93. The polynucleotide according to claim 83, further comprising an exon 1 sequence, an exon 2b sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence.

94. The polynucleotide according to claim 93, comprising the sequence set forth in any one of SEQ ID NO: 11-15.

95. The polynucleotide according to claim 83, further comprising an exon 1 sequence, an exon 2b sequence, an exon 3 sequence and an exon 4 sequence.

96. The polynucleotide according to claim 95, comprising the sequence set forth in any one of SEQ ID NO: 16-20.

97. The polynucleotide according to claim 83, further comprising an exon 1 sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence.

98. The polynucleotide according to claim 97, comprising the sequence set forth in any one of SEQ ID NO: 21-25.

99. The polynucleotide according to claim 83, further comprising an exon 1 sequence, an exon 3 sequence and an exon 4 sequence.

100. The polynucleotide according to claim 99, comprising the sequence set forth in any one of SEQ ID NO: 31-35.

101. An isolated polynucleotide that consists essentially of at least a portion of a nucleic acid sequence selected from SEQ ID NO: 7, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 7, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 7 under at least high stringency conditions.

102. The polynucleotide according to claim 101, further comprising an exon Ia sequence.

103. The polynucleotide according to claim 101, further comprising an exon Ib sequence.

104. The polynucleotide according to claim 101, further comprising an exon Ic sequence.

105. The polynucleotide according to claim 101, further comprising an exon Id sequence.

106. The polynucleotide according to claim 101, further comprising an exon Ie sequence.

107. The polynucleotide according to claim 101, further comprising an exon 2a sequence.

108. The polynucleotide according to claim 101, further comprising an exon 2c sequence.

109. The polynucleotide according to claim 101, further comprising an exon 3 sequence.

110. The polynucleotide according to claim 101, further comprising an exon 4 sequence.

111. The polynucleotide according to claim 101, further comprising an exon 1 sequence, an exon 2a sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence.

112. The polynucleotide according to claim 101, comprising the sequence set forth in any one of SEQ ID NO: 11-15.

113. The polynucleotide according to claim 101, further comprising an exon 1 sequence, an exon 2a sequence, an exon 3 sequence and an exon 4 sequence.

114. The polynucleotide according to claim 113, comprising the sequence set forth in any one of SEQ ID NO: 16-20.

115. The polynucleotide according to claim 101, further comprising an exon 1 sequence, an exon 2c sequence, an exon 3 sequence and an exon 4 sequence.

116. The polynucleotide according to claim 115, comprising the sequence set forth in any one of SEQ ID NO: 26-30.

117. The polynucleotide according to claim 101, further comprising an exon 1 sequence, an exon 3 sequence and an exon 4 sequence.

118. The polynucleotide according to claim 117, comprising the sequence set forth in any one of SEQ ID NO: 35-40.

119. An isolated polynucleotide that consists essentially of an exon 1 nucleotide sequence selected from: (1) an exon Ib sequence as set forth in SEQ ID NO: 2, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 2, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 2 under at least high stringency conditions; (2) an exon Ic sequence as set forth in SEQ ID NO: 3, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 3, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 3 under at least high stringency conditions; (3) an exon Id sequence as set forth in SEQ ID NO: 4, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 4, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 4 under at least high stringency conditions; and (4) an exon Ie sequence as set forth in SEQ ID NO: 5, or a sequence that displays at least 90% sequence identity to the sequence set forth in SEQ ID NO: 5, or a sequence that hybridizes to the complement of the sequence set forth in SEQ ID NO: 5 under at least high stringency conditions.

120. The polynucleotide according to claim 119, further comprising an exon 2a sequence.

121. The polynucleotide according to claim 119, further comprising an exon 2b sequence.

122. The polynucleotide according to claim 119, further comprising an exon 2c sequence.

123. The polynucleotide according to claim 119, further comprising an exon 3 sequence.

124. The polynucleotide according to claim 119, further comprising an exon 4 sequence.

125. The polynucleotide according to claim 119, further comprising downstream of the exon 1 sequence an exon 2a sequence, an exon 2b sequence, and exon 2c sequence, an exon 3 sequence and an exon 4 sequence.

126. A chimeric nucleic acid construct comprising the polynucleotide of any one of claims 83 to 125, which is operably connected to a regulatory element that is operable in a host cell.

127. An isolated host cell comprising the nucleic acid construct of claim 126.

128. A probe for interrogating nucleic acid for the presence of the polynucleotide of any one of claims 83 to 125, the probe consisting essentially of a nucleotide sequence that hybridizes under at least high stringency conditions to the polynucleotide of any one of claims 83 to 125.

129. The probe of claim 128, which consists essentially of the sequence set forth in any one of SEQ ID NO: 46 to 52.

130. A kit for diagnosing the presence or risk of development of prostate cancer, the kit comprising one or more probes as defined in claim 128 or claim 129.

131. The kit according to claim 130, further comprising reagents and instructions for use in the method according to any one of claims 1 to 82.

132. A method for treating or preventing prostate cancer in a subject, the method comprising: detecting in the subject expression or overexpression of a polynucleotide as defined in any preceding claim, and administering to the subject at least one therapy that treats or ameliorates the symptoms or reverses or inhibits the development or progression of the prostate cancer in the subject.