WO2012071665A1 - Prognostic markers of inherited variations in the ugt2b genes for prostate cancer recurrence - Google Patents

Abstract

The present application discloses that reduced copy number variations (CNVs) in UGT2B17 and UGT2B28 were significantly associated with bRFS while no significant association was observed for additional UGT variants tested. The co-occurrence of two or more deletions of UGT2B17 and UGT2B28 genes resulted in an HR of 1.377 (95% CI [1.105; 1.716]; p=0.004) for Caucasians and 2.151 (95% CI [1.239; 3.372]; p=0.006) for Asians. In all studied men, risk of BCR was 1.510; 95% CI [1.178 - 1.950]; p=0.001 ). The addition of the UGT2B CNV status in the D'Amico risk classification further enhanced the risk prediction mainly for the low and intermediate risk categories (Log-rank test, p=0.022 and 0.011, respectively). In addition, after adjusting for all clinicopathologic risk factors, we found a strong association between the risk of BCR and 7 SNPs in SRD5A genes. The combination of 2 SNPs respectively in SRD5A1 and SRD5A2 were highly favourable, reducing drastically the risk of BCR for carriers of 3-4 alleles (HR=0.34; 95% CN0.18- 0.64; P=9x10"4). Other variations in the SRD5A2 gene were associated with an increased rate of BCR, namely the coding SNPs rs523349 with a HR of 2.12 (95% CI, 1.21 -3.75; P=0.009) and reaching 4.97 when combined with deleted copies of UGT2B genes (95% CI, 2.38-10.36; P=0.00002). Further, BCR-free survival rate was significantly reduced to 27% in patients with unfavourable genotypes compared to 75% for patients with favourable genotypes (P=7x10^-6). This study is the first to recognize CNV in genes at the end of the androgenic signal as significant and independent predictors of PCa recurrence after prostatectomy. In addition, inherited polymorphisms in the SRD5A and UGT2B genes are independent predictors of biochemical recurrence after radical prostatectomy.

Description

Prognostic Markers of Inherited variations in the UGT2B genes for Prostate

Cancer Recurrence

Cross-reference to related applications [0001] This application claims priority from US provisional patent application 61/419,057 filed on December 2, 2010, the content of which is incorporated by reference in its entirety.

Field of the invention

[0002] The present invention relates to the use of markers to identify relevant patterns of inherited variation in genes involved in sex-steroid metabolism, and more particularly the UGT2B gene, optionally with the SRD5A gene, for use in prognosticating or predicting the recurrence of prostate cancer.

Background of the invention

[0003] Prostate cancer (PCa), an androgen- and estrogen-related cancer, is the sixth most common cancer in the world and the second leading cause of cancer death in North American men. The specific mortality of PCa is directly related to both the stage at time of diagnosis and age at onset. Of the several known risk factors, the most significant are age, ethnicity, genetic and dietary factors. In the early stages of carcinogenesis, androgens are centrally implicated in the development and progression of this hormone- dependent cancer. However, an association between plasma androgen levels and PCa susceptibility has not been consistently observed across studies.

[0004] Currently, PCa recurrence risk is assessed based on clinical stage, biochemical prostate-specific antigen (PSA) level, and Gleason scores. Patients with localized (clinical stage T1 -T2) and locally advanced (T3) PCa are frequently treated with radical prostatectomy (RP), a potentially curative procedure. However, it is estimated that over 30% of men undergoing RP will have disease relapse, also referred to as biochemical recurrence (BCR) as the first clinical indication of rising serum level of PSA .

[0005] Currently, the Tumor/Nodes/Metastasis (TNM) staging system, the Gleason score, and pre-treatment serum prostate-specific antigen (PSA) are the most important factors influencing both the likelihood of more extensive disease and the probability of

subsequent relapse following RP. Indeed, these tools include both nomograms and risk tables incorporating clinical variables that can predict, although still imperfectly, the likelihood of tumor recurrence and provide crucial prognosis information to guide clinicians in their therapeutic decisions. The risk of disease progression greatly differs between individuals and the heterogeneity in clinical behaviour further emphasizes the need to find novel markers of progression. Even if cases of PCa are considered localized at the time of diagnosis, the rate of BCR after RP is still significant and occur most often in the 5 years after surgery. The persistence of tumor cells in a state of either complete or near dormancy prior to metastatic progression is likely accountable for disease recurrence while these residual cells are most probably responsive to hormones.

[0006] However, in two recently published large-scale population trials, PSA screening was shown to have little impact on decreasing cancer-related mortality, and was accompanied by a risk of overdiagnosis, overtreatment, and increased complications. Hence, the heterogeneity observed in clinical behavior emphasizes the need to identify additional tools suitable for use in clinical practice, such as molecular biomarkers.

[0007] Androgen hormones, such as testosterone (T) and 5a-dihydrotestosterone (DHT), have been clearly implicated in development of PCa. Circulating T, secreted by the testis, and adrenal steroid precursors are among factors influencing androgen levels in the prostate. Indeed, the conversion of T by 5a-reductases (SRD5A1 and SRD5A2) leads to DHT, the more potent androgen receptor (AR) endogenous agonist in target cells.

SRD5A2 is the major 5a-reductase enzyme expressed in the prostate compared to SRD5A1¹. However, while the expression of SRD5A2 decreases in prostate cancer cells, SRD5A1 is increased in tumoral tissues^1"3. This imbalance in the expression of SRD5A genes in PCa tumors illustrates the complex relation between 5a-reductases, DHT synthesis and PCa progression. In concert with SRD5A genes, the active androgen level in target cells is controlled by steroid metabolizing UDP-glucuronosyltransferases 2B genes (UGT2B). These phase II metabolism enzymes conjugate androgens, and thus contribute to the irreversible transformation of steroids into biologically inactive

glucuronide metabolites^4,5,6. Consequently, the intracellular hormonal level is determined by the balance between SRD5A and UGT2B enzymatic activities. [0008] Androgen deprivation therapy (ADT) is the standard of care for metastatic PCa and is also used to treat asymptomatic patients with PSA recurrence after failed primary therapy (RP), further reinforcing the initial androgen dependency of these cells.

Finasteride, a 5a-reductase type 2 inhibitor, and Dutasteride, a dual 5a-reductase inhibitor currently used in the clinic, have been recently shown to be effective chemopreventive medications reducing by almost 25% the risk of PCa incidence^{7 8}. Despite this well recognized hormonal dependence of prostate cancer cells in the early cancer stage, very few studies have investigated the associations between polymorphisms in the androgen biosynthesis pathway and clinical outcome after surgical procedure^8"14. To date, common polymorphisms such as those in sex-steroids biosynthesis pathways have been extensively studied in relation to risk of PCa^15"19. However, almost all of these studies did not address the association between polymorphisms in genes regulating hormonal exposure with PCa recurrence and survival, and were not designed to do so. Long-term longitudinal studies are thus still required to systematically evaluate the impact of a patient's genetic profile on risk of recurrence.

[0009] One of the most significant challenges in oncology remains our inability to predict PCa disease recurrence and clinical outcome due to a lack of key prognostic markers. The prediction of recurrence following RP is clearly important for personalized treatments and follow-up strategies due to striking heterogeneity in prostate cancer clinical behaviour. Our work support that inherited variations in sex-steroid biosynthesis enzymes are relevant candidates as novel predictive markers.

[0010] Over the last several decades, a number of studies have addressed the role of inherited variations in hormone-related genes and the associated risk of developing prostate cancer.^15,16,20,21 However, to our knowledge, only two studies have looked at the prognostic value of hormone-related gene polymorphisms in PCa progression and mortality.^{11 ,22} In the first study, Lindstrom and colleagues studied the role of germline variations in AR genes and the biosynthetic genes CYP17 and SRD5A2, and suggested that polymorphisms in the AR genes affect hormonal treatment and ultimately PCa death.¹¹ More recently, overexpression of the androgen/estrogen inactivating enzyme HSD17B4 was associated with a poor clinical outcome.²² Despite the fundamental role of androgens in PCa development, very scarce data are available regarding the

consequence of genetic variations in the steroidogenic pathway and the associated risk of PCa progression following treatment, particularly for early stage disease. No other predictive or prognostic markers have been identified in the steroidogenic pathway, although several prognostic biomarkers have been identified in other cellular pathways, including cell cycle genes, tumor suppressor genes, cytokines, and transcription factors. [0011] UDP-glucuronosyltransferases (UGTs) are conjugating enzymes that, in concert with other steroidogenesis enzymes, contribute to the maintenance of intracellular levels of sex-steroid hormones in target cells. This enzymatic process generates glucuronide derivatives that are biologically inactive and are subsequently excreted in bile or urine. UGTs are expressed in almost all tissues, including hormone target organs such as the prostate. Five UGT isoforms are mainly responsible for inactivating androgens and estrogens. The UGT1A1 isoform is primarily involved in the glucuronidation of estradiol, whereas the UGT2B class of enzymes (namely UGT2B7, UGT2B15, UGT2B17, and UGT2B28) is mainly involved in androgen catabolism in hormone-dependent cells.

Common polymorphisms in two of these genes (UGT2B15 and UGT2B17) have been associated with prostate cancer risk in some but not all studies.^18,23"30 Interestingly, copy number variations (CNV) of the major UGT2B17 and UGT2B28 androgen metabolizing genes have been reported, and more than 50% of Caucasians and 75% of Asians have a reduced copy number in at least one of these genes.³¹ However, the impact of common germline polymorphic variations or deletions in UGTs has not yet been addressed in PCa progression and mortality.

Summary of the invention

[0012] In the present invention, functional genetic variations in each of the following sex- steroid metabolizing genes UGT1A1 , UGT2B7, UGT2B15, UGT2B17, and UGT2B28, were studied to identify potential molecular biomarkers that are associated with an increased risk of biochemical PSA recurrence after prostatectomy. These UGT candidates were selected due to their prostate-specific role in steroid catabolism. Four populations of patients (three Caucasians and 1 Asian) comprising a total of 1161 men with clinically localized and locally advanced prostate cancer were studied.

[0013] The present invention identifies germline polymorphic CNV events in UGT2B17 and UGT2B28 that are significantly associated with biochemical recurrence in clinically localized PCa after surgical treatment. These deletions are linked to altered levels of circulating sex steroids, supporting the hypothesis that reduced androgen glucuronidation by UGT-mediated inactivation leads to an increased PSA recurrence. Our data indicates that co-occurrence of deleted copies of the UGT2B17 and UGT2B28 genes are important predictors for PSA recurrence independently of the well-established clinico-pathological markers; Gleason score, PSA values and stages at the time of diagnosis.

[0014] To further improve the prognosis significance of inherited variation in the sex- steroid metabolizing genes, genetic variation in the androgen biosynthesis SRD5A genes were combined with CNV in the androgen inactivating UGT2B genes. Together, these genes may alter systemic hormone bioavailability and influence the tumoral

microenvironment exposure to hormones, and thus could modify the risk of PCa recurrence after RP. This is based on the fact that both classes of genes have a well characterized physiological role in DHT homeostasis, are associated with PCa risk and, more recently with cancer progression ¹². ³.¹⁸·²³.²⁴·²⁶.²⁷·²⁹·³²·³³.⁴⁶, SRD5A genes are also well recognized molecular targets in PCa prevention trials^7,35. In one aspect of the invention, the detailed genetic analyses of the SRD5A1 and SRD5A2 genes in relation to PCa progression are presented. The association between the known SRD5A2 V89L polymorphism and BCR as proposed by other groups^12,13 is also described. In one particular aspect, the association between inherited genetic alterations in androgen metabolizing genes and the risk of relapse was determined after RP as the sole initial curative intent. In a particular embodiment, the clinical consequence of simultaneous occurrence of variations in the androgen biosynthetic SRD5A genes and in UGT2B sex- steroid inactivating genes is used to improve the statistical significance of the method for predicting the likelihood of a human subject to develop PCa recurrence or the risk of relapse after RP. [0015] According to a first aspect of the present invention, there is provided a biomarker for predicting the likelihood of prostate cancer development, its progression or recurrence in a human subject, comprising the identification of at least one copy number variation(s) in at least one of a UGT2B gene.

[0016] According to a further aspect, the invention provides the identification of CNV(s) in at least one of a UGT2B gene of a human subject for predicting the likelihood of prostate cancer, its progression or recurrence in said human subject. [0017] There is therefore provided an in-vitro method for predicting risk for PCa in a human subject, said method comprising the steps of: a) obtaining a nucleic acid from a non-tumor or tumor sample from said human subject; and b) determining an individual's genetic variation (or haplotypes, CNV or deletion) in a UGT2B gene; whereby the identification of at least one genetic variation in said UGT2B gene in said subject's nucleic acid is an indication that said subject has an increased risk of developing prostate cancer or of experiencing progression thereof. Particularly, UGT2B risk alleles may be defined as one deletion of UGT2B28 or UGT2B17 and/or two or more deletions in UGT genes.

[0018] The invention further provides the method as defined herein wherein said identification of genetic variation in said UGT2B17 or UGT2B28 comprises identifying at least one CNV in said nucleic acids, said CNV being selected from the group consisting of: rs3100645; and rs1 1249532; and associated reference sequences thereof.

[0019] The method of the invention further comprises the identification of the cooccurrence of a genetic variation (or haplotype) in SRD5A1 or SRD5A2 gene; whereby the co-occurrence of at least one genetic variation in UGT and SRD5A1 or SRD5A2 in said subject's nucleic acid is an indication that said subject has a further increased likelihood that the PCa will recur.

[0020] The invention further provides the method as defined herein wherein said identification of genetic variation in said SRD5A1 or SRD5A2 comprises identifying at least one single nucleotide polymorphism (SNP) in said nucleic acids, said SNP being selected from the group consisting of: rs166050; rs2208532; rs523349; rs4952197;

rs518673, rs12470143 and rs676033; and associated reference sequences thereof.

[0021] The present invention also provides a method for adapting a course of treatment of prostate cancer in a human subject, comprising the steps of: a) providing a prognosis or predicting the likelihood of a human subject to develop prostate cancer recurrence in accordance with the method as defined herein; and b) adapting a course of treatment according to whether said subject has an increased likelihood that the cancer will recur. Particularly, adaptation of course of treatment is realized after the subject has undergone radical prostatectomy. [0022] The invention further provides a kit for predicting the likelihood of a human subject to develop prostate cancer recurrence by detecting a CNV in a nucleic acid sample, the kit comprising reagents for determining the individual's genetic variations (or haplotypes) in UGT2B17 and UGT2B28 gene.

[0023] Particularly, the kit comprises a reagent for detecting a reference sequence selected from the group consisting of: rs3100645; and rs1 1249532, and associated reference sequences thereof.

[0024] Particularly, the kit further comprises reagents for determining the individual's genetic variations (or haplotypes) in SRD5A1 or SRD5A2 gene. Particularly, the kit further comprises reagents for detecting a SNP in a reference sequence selected from the group consisting of: rs518673; rs166050; rs12470143; rs2208532; rs523349; rs4952197 and rs676033 or their associated SNPs.

Detailed description of the invention

Description of the figures

[0025] Figure 1. PCR amplifications of the UGT2B17 and UGT2B28 genes.

[0026] Figure 2. Kaplan-Meier curves of Biochemical Recurrence-Free Survival (bRFS) for copy-number variations of UGT2B genes.

[0027] Figure 3. Kaplan-Meier curves of bRFS for co-occurrence of UGT2B risk allele. In Caucasians, UGT2B risk alleles were defined as 1 deletion of UGT2B28 and/or two or more deletions in UGT genes. In Asians, UGT2B risk alleles were defined as two or more deletions in UGT genes. [0028] Figure 4. Association analysis of common UGT2B17 and UGT2B28 gene deletions and biochemical failure in additional cohorts of PCa cases. Odds ratios and confidence intervals were calculated to combine data from the 1 ,161 PCa cases. UGT2B risk alleles were defined as 1 deletion of UGT2B28 and/or two or more deletions in UGT genes. [0029] Figure 5. Prognostic impact of inclusion of genetic markers, UGT2B17 and UGT2B28 gene deletions, in the evaluation of the D'Amico risk classification of PCa recurrence after RP. [0030] Figure 6. Kaplan-Meier Biochemical Recurrence-Free Survival Analysis for other UGT genes.

[0031] Figure 7. Risk of recurrence associated with known clinical and pathological prognostic variables (A) and SRD5A genes (B). Boxes represent hazard ratios (HR) and their 95% CI. PSA categories are in ng/ml. Reference categories (HR: 1.00) are: PSA at diagnosis < 10ng/ml, pG < 6, and pT < T2b. Genetic linkage between htSNPs tested for each SRD5A gene is represented in the triangles on the left in panel B.

[0032] Figure 8. Kaplan-Meier estimates of recurrence-free survival for A) SRD5A2, B) SRD5A 1 and C) protective htSNPs in both SRD5A genes. Log rank (LR) P values are shown in each frame.

[0033] Figure 9. Risk of recurrence associated with htSNPs in A) SRD5A1 and B)

SRD5A2 genes stratified by status for UGT2B risk alleles. Boxes represent hazard ratios (HR) and their 95% CI in multivariate analysis. Black boxes were analyzed only for SRD5A genotypes, white boxes are for SRD5A minor alleles + no UGT2B risk alleles, and grey boxes are for SRD5A minor alleles + UGT2B risk alleles. Boxes indicate HR and error bars indicate the 95% confidence intervals. The reference category is set at one. Models used for multivariate analysis are indicated in Table 13.

[0034] Figure 10. Kaplan-Meier curves of biochemical recurrence-free survival for carriers of protective alleles of both SRD5A genes and of UGT2B risk alleles. Log-rank P values (LR) are shown in each frame. Risk alleles for UGT2B are defined as one deleted copy of UGT2B28 or >2 deleted copies of UGT2B17 and UGT2B28. 0= no protective SRD5A alleles; 1 -2= 1 or 2 protective SRD5A alleles and 3-4= 3 or 4 protective SRD5A alleles.

Definitions and abbreviations

[0035] ADT: androgen-deprivation therapy; AR: androgen receptor; BCR: biochemical recurrence; bRFS: biochemical recurrence-free survival, CNV: copy number variation; DHT: 5a-dihydrotestoterone; HR: hazard ratio; htSNP: haplotype-tagging SNP; MAF: minor allele frequencies; PCa: prostate cancer; PSA: prostate-specific antigen; SRD5A1 : 5a-reductase type 1 ; SRD5A2: 5a-reductase type 2; RP: radical prostatectomy; SNP: Single nucleotide polymorphism; T: Testosterone; TNM: Tumor/Nodes/Metastasis staging system; UGT: UDP-glucuronosyltransferase. [0036] The term "identify" as used herein, means the act of searching for and finding a specific marker. Other terms that may be used for the same purpose are: "detect", "locate", "expose", "observe", "recognize", "uncover" or "reveal".

[0037] The term "development" or "developing" when used in the context of cancer means the development, progression, recurrence, biochemical recurrence, death from or metastasis of such a cancer.

[0038] The term 'sample" as used herein, means a patient or subject's nucleic acid- containing sample originating indistinctly from a tumor or a non-tumor sample. The subject's non-tumor sample can be selected from: tissue, or biological fluid. Particularly, the tissue can be selected from the group consisting of: skin, lymph node, hair and buccal smear. Alternatively, the biological fluid can be selected from the group consisting of: sputum, saliva, blood, serum, urine, semen and plasma. Alternatively, the non-tumor or tumor sample may originate from a biopsy.

[0039] The terms "genetic variation" or "haplotype" used herein in conjunction with UGTs mean "Copy-number variations" (CNVs), e.g. a form of structural variation consisting in alterations of the DNA of a genome that results in the cell having an abnormal number of copies of one or more sections of the DNA. CNVs correspond to relatively large regions of the genome that have been deleted (fewer than the normal number) or duplicated (more than the normal number) on certain chromosomes. For example, the chromosome that normally has sections in order as A-B-C-D might instead have sections A-B-C-C-D (a duplication of "C") or A-B-D (a deletion of "C"). When used in conjunction with SDR5A gene, the terms "genetic variation" or "haplotype" refer to single-nucleotide polymorphisms (SNPs), which are DNA sequence variations occurring when a single nucleotide base— A, T, C or G— in the genome, or other shared sequence, differs between members of a biological species or paired chromosomes in an individual.

Detailed description of particular embodiments

Genetic variation status, copy number variation and reference sequence.

[0040] In accordance with particular aspects of the present invention, there is provided an in-vitro method for predicting the risk for PCa in a human subject, said method comprising the steps of: a) obtaining a nucleic acid from a non-tumor or tumor sample from said human subject; and b) determining the individual's genetic variation status such as for example a copy number variation in UGT2B17 gene and/or UGT2B28 gene; whereby the determination of at least one genetic variation in one of the UGT2B17 or UGT2B28 gene is an indication that said subject has an increased likelihood that the prostate cancer will recur.

[0041] The invention further provides the method as defined herein wherein said identification of genetic variation status in said UGT2B17 or UGT2B28 comprises identifying at least one copy number variation (CNV) in said nucleic acids, said CNV being selected from the group consisting of: rs3100645; and rs1 1249532. [0042] Particularly, the genetic variation in UGT2B17 is a copy number variation consisting of one or two deletions. Still, particularly, the genetic variation in UGT2B28 is a copy number variation consisting of one or two deletions.

[0043] Still, more particularly, the method of the invention comprises the identification of the co-occurrence of the genetic variations in UGT2B17 and UGT2B28 genes, e.g. when both UGT2B17 and UGT2B28 genes harbor at least one CNV selected from the group consisting of: rs3100645; and rs 1249532. Particularly, each of UGT2B17 and UGT2B28 can harbor one or two deletions.

Detection of co-occurrence with other markers

[0044] The method of the invention further comprises the identification of the co- occurrence of a genetic variation in SRD5A1 or SRD5A2 gene; whereby the cooccurrence of at least one genetic variation in UGT2B and either SRD5A1 or SRD5A2 or both in said subject's nucleic acid is an indication that said subject has a further increased likelihood that the prostate cancer will recur.

[0045] The invention further provides the method as defined herein wherein said identification of genetic variation in said SRD5A1 or SRD5A2 comprises identifying at least one single nucleotide polymorphism (SNP) in said nucleic acids, said SNP being selected from the group consisting of: rs166050; rs2208532; rs523349; rs4952197;

rs518673, rs 12470143 and rs676033. Sample

[0046] The invention also provides the method as defined herein, wherein the subject's nucleic acid-containing sample is a tumor or a non-tumor sample. Particularly, the subject's non-tumor sample is selected from: tissue, or biological fluid. Particularly, the tissue is selected from the group consisting of: skin, lymph node, hair and buccal smear. Alternatively, the biological fluid is selected from the group consisting of: sputum, saliva, blood, serum, urine, semen and plasma. Alternatively, the non-tumor or tumor sample originates from a biopsy.

Methodology

[0047] The invention also provides the method as defined herein wherein in step a), the nucleic acid is DNA or RNA. Particularly, the DNA and the marker is detected with the use of a nucleic acid primer. More particularly, the DNA is amplified by PCR.

[0048] Particularly, the genetic variation of DNA or RNA is detected with the use of a methodology selected from the group consisting of: direct sequencing, pyrosequencing, massively parallel sequencing, high-throughput sequencing, high performance liquid chromatography (HPLC) fragment analysis, mass spectrometry, branch DNA and capillary electrophoresis. Alternatively, the DNA or RNA of the marker is detected with the use of a nucleic acid primer and probe combination. Particularly, the DNA or RNA is amplified prior to incubation with the probe, such as by PCR. More particularly, amplified PCR products and the genetic variation are analyzed by mass spectrometry.

Treatment regimen.

[0049] The present invention also provides for a method for adapting a course of treatment of prostate cancer in a human subject, comprising the steps of: a) predicting the likelihood of prostate cancer progression or recurrence in a human subject as defined herein; and b) adapting a course of treatment according to whether said subject has an increased or decreased likelihood that the cancer will progress, or recur.

[0050] Particularly, when the likelihood of recurrence is increased, the subject is prescribed at least one of: hormone therapy, chemotherapy, surgery, radiotherapy, brachytherapy or active surveillance (watchful waiting). More particularly, steps a) and b) are carried out after the subject has undergone radical prostatectomy. [0051] The invention also provides the method as defined herein, wherein, when the likelihood of recurrence is increased, the subject is prescribed inducer (or inductive) UGT2B therapy and/or 5a-reductase inhibitors therapy.

Kit and reagents

[0052] Furthermore, the present invention provides for a kit for predicting the likelihood of a human subject to develop prostate cancer or a recurrence thereof, said kit comprising reagents for determining the individual's genetic variations (or haplotypes) in the UGT2B gene. Particularly, the kit comprises reagents for detecting a CNV in a reference sequence selected from the group consisting of: rs3100645 and/or rs1 1249532; and associated reference sequences thereof.

[0053] The invention also provides the kit as defined herein, comprising a PCR primer, wherein the primer is selected from the group consisting of: SEQ ID Nos. 1 , 2, 7 and 8. Additionally, the kit may comprise a PCR probe, wherein the probe hybridizes with a sequence selected from rs3100645 and/or rs1 1249532; and associated reference sequences thereof.

[0054] Particularly, the kit further comprises reagents for determining the individual's genetic variations (or haplotypes, SNP) in SRD5A1 and/or SRD5A2 genes, such as reagents for detecting a SNP in a reference sequence selected from the group consisting of: rs518673; rs166050; rs12470143; rs2208532; rs523349; rs4952197 and rs676033 or their associated SNPs.

[0055] The invention further provides the kit as defined herein, comprising PCR primer- probe set for determining the individual's SNPs in SRD5A1 and/or SRD5A2 genes, wherein the primer is selected from the group consisting of: SEQ ID Nos. 12 to 50; and the probe is selected from the group consisting of: SEQ ID Nos. 51 to 69. Example 1 - Copy number variations in the UGT2B genes

Patients and methods

Description of Study Cohorts.

[0056] The copy number variations in the UGT2B genes were studied in 1 161 unrelated men obtained from four independent cohorts; cohort A was composed of 526 Caucasian patients with localized prostate cancer; cohort B comprised 213 Caucasian patients with locally advanced disease, cohort C comprised 320 Asian patients with localized prostate cancer and cohort D was composed of 102 Caucasian Slovenian men (Table 2). Cohorts A and B patients underwent open radical prostatectomy for clinically localized and locally advanced adenocarcinoma of the prostate at the CHUQ - Hotel-Dieu de Quebec hospital (Quebec, Qc, Canada) between 1982 and 2002. Cohorts C and D have been described elsewhere.^8,36 Each participant gave written informed consent before surgery for subsequent genetic analyses. The local research ethical committee approved the research protocol. Detailed clinical information was obtained retrospectively from medical records. All patients were followed postoperatively with serial PSA measurements.

Definitions.

[0057] The primary outcome variable was biochemical recurrence-free survival (bRFS), also called 'PSA failure' or 'biochemical relapse'. In Caucasians, bRFS was defined as (1 ) two consecutive PSA values > 0.3 pg/L, (2) one PSA value≥ 0.3 pg/L followed by hormonotherapy (androgen deprivation therapy or ADT) or radiation therapy, and (3) a single last-recorded PSA value > 0.3 pg/L after prostatectomy. bRFS was defined as the period of time elapsed between the surgical procedure and the date of PSA failure. In cohorts C and D, the bRFS was defined as previously described. ^{8 36}

Genotyping.

[0058] For cohorts A and B, peripheral blood was collected on the morning of a preoperative ambulatory clinic visit. All samples were kept frozen at -80°C until the time of study. Genomic DNA was extracted using a QIAamp DNA Blood mini kit (Qiagen Inc, Mississauga, Ontario, Canada) and stored at -80°C. For cohorts C and D, DNA was prepared as previously described. ^{8 36} [0059] The presence of at least one copy of UGT2B17 and UGT2B28 is first assessed by PCR amplifications of the exon 1 of each gene and the corresponding deletion site, using PCR conditions and specific primers as previously described ³¹.

[0060] Common amplification steps used are: one heating step at 95° C for 30 sec, 40 cycles of denaturing at 95°C, annealing and elongation at 72°C and a single final extension step of 7 min at 72°C (see Figure 1 and Table 1 ). [0061] Amplification of the exon 6 can be performed to confirm the absence of the studied gene (UGT2B17 and UGT2B28) if deemed necessary, in case of uncertainties only. With these genotyping strategies, we were able to detect precisely the number of deletions (from 0 to 4).³¹ [0062] For UGT1A1 (rs34815109), UGT2B15 (rs1902023), and UGT2B7 (rs7439366), after PCR amplification (for primers and amplification strategies, see ^{31 ,37}), products were purified and sequenced with an ABI 3730x1 genetic analyzer (Applied Biosystems, Foster City, CA, USA) and assessed only in Cohort A. The sequences were analyzed using Staden preGap and Gap4 programs (Open Source Technology Group,

http://staden.sourceforge.net/) to identify the variant sequence. PCR amplicons were sequenced, and amplicons resulting in ambiguous sequencing chromatograms were systematically re-amplified and re-sequenced.

Statistical Analysis

[0063] Based on our sample size, we were able to detect a hazard ratio of 1.50, for a minor allele with frequency of 5%, with over 80% statistical power. All genes were tested for consistency with Hardy-Weinberg equilibrium using chi-square analysis. Each gene was treated as a categorical variable, with a common (reference) homozygote, a heterozygote, and a rare homozygote. A rare homozygote was combined with a heterozygote if the frequency of the rare homozygote was below 0.10. For UGT2B17, it was previously established that deletion of one allele is not associated with significantly lower intraprostatic mRNA levels compared to individuals homozygous for the insertion allele (ins/ins)³⁵. Therefore, individuals with one deletion allele of UGT2B17 were combined with subjects with no deletion. For UGT2B28, the rare del/del genotype was combined with ins/del heterozygote because the frequency of the null genotype was below 0.10. Initial PSA level, Gleason score at biopsy, and tumor clinical stages were treated as categorical variables using the well-recognized D'Amico risk classification. In cohorts C and D, the pathological stage was used for multivariate analysis because tumor clinical stage was not available.

[0064] Kaplan-Meier analysis for bRFS analysis was performed to compare genotypes using the log-rank test followed by a pairwise multiple comparison using the Sidak multiple-comparison method. Results that were significantly associated with bRFS in univariate analysis were then evaluated in a multivariate Cox regression analysis. We included the covariates included in Table 2 in the models and UGT2B17 and UGT2B28 genetic status. The proportional hazard assumption of Cox models was verified graphically by log-minus-log vs. log (time) plots for each variable. Kaplan-Meier curves and univariate analysis for bRFS were also performed for CNVs of UGT2B17 and

UGT2B28. Haplotype analysis was performed for cohort A only. After this initial screen, the other genetic variables were not further studied in other cohorts due to lack of association. Clinical variables were compared across genotypes using the chi-square test, Fisher's exact test evaluated exactly, or the Monte Carlo approximation, as appropriate. A p value of less than 0.05 was considered statistically significant, and all tests were two- sided. All statistical analyses were performed using SAS® Statistical Software version 9.2 (SAS Institute, Cary, NC, USA).

RESULTS

[0065] Patient's characteristics are presented in Table 2. Overall, 130 patients (24.7%) experienced PSA failure in cohort A, 132 (62%) in cohort B, 1 16 (36.3%) in cohort C and 34 (33%) in cohort D.

[0066] Preoperative PSA values and pathological biopsy Gleason scores were both associated with bRFS. Men with PSA values >20 were associated with an HR of 2.120 (95% [1.542; 2.913], p = 0.000004) for bRFS in multivariate analysis (Table 4), whereas a Gleason score >8 is associated with an HR of 2.773 (95% CI [2.005; 3.835], p < 0.0001 ) and clinical T stage with an HR of 2.046 (95% CI [1.264; 3.313], p < 0.004); similar to previous reports. ^38,39 Reduced CNVs in UGT2B17 and UGT2B28 were significantly associated with bRFS (Table 3 and Figure 2) while no significant association was observed for additional UGT variants tested in cohort A (Table 5). Haplotype analyses of the UGT2B locus in this cohort also revealed that haplotypes 3 and 5, containing UGT2B deletions, were associated with bRFS (Table 6). Based on these initial results, additional cohorts were tested. The distributions of genotype frequencies are shown in Table 7 and are consistent with previous reports.^{26,31 ,40,41}

[0067] Figure 2 displays the Kaplan-Meier curves of bRFS for UGT2B17 and UGT2B28 genes for all studied patients for whom this analysis was achievable (cohorts A, B and C). In cohorts A and B, multivariate analyses indicated that individuals with one UGT2B28 or two UGT2B17 deleted alleles had an increased likelihood of PSA failure with HR values of 1 .390 (95% CI [1.064; 1.815], p = 0.016) and 1.385 (95% CI [0.961 ; 1.997], p = 0.08), respectively (Table 3). Individuals of Asian Ancestry (cohort C) were included in our study because they have the highest reported frequencies of UGT2B deletions. ⁴¹ Comparison of UGT2B17 CNVs minor allele frequencies (MAF) revealed that the UGT2B17 deletion genotype prevailed in this population with a MAF of 0.73, compared to 0.27 in Caucasians (Table 7). In this population, 57% of individuals (n=185) have a UGT2B17 null genotype and, therefore, constitute the most common genotype in this ethnic group. For UGT2B28, the MAF is 0.15 in Asians which is similar to the frequency of 0.13 observed in

Caucasians. Six subjects with the combined UGT2B17 and UGT2B28 null genotypes were identified, indicating for the first time that concurrent deletions in these UGT2B genes are rare but not lethal. In the population of Asians, data revealed that two deleted copies of the UGT2B17 gene is associated with biochemical failure with an HR of 1 .791 (95% CI [1.107; 2.898]; p=0.018). Patients genotyped with two or more deletions of UGT2B17 and UGT2B28 genes have an increased risk of BCR with HR of 2.151 (95% CI [1.239; 3.372]; p=0.006) for bRFS (Figure 3 and Table 3). Multivariate analysis of all 1161 patients lead to an OR of 1.510 (95% CI [1 .178 - 1.950]; p=0.001 ) associated with the combined effect of UGT2B deletions (Figure 4). These results support that UGT2B17 and UGT2B28 CNVs are determinants of PSA failure after prostatectomy. [0068] To further demonstrate the clinical relevance of UGT2B deletions on bRFS, we used the D'Amico risk classification and further stratified each category for the status for UGT2B risk alleles in Caucasians (cohorts A and B) (Figure 5). The addition of the information regarding CNV status led to an improved risk prediction, particularly for the low and intermediate risk categories (Log-rank (LR) test, p=0.022 and 0.01 1 , respectively) (Figure 5). This further supports that genetic deletions in the UGT2B pathway are independent predictors of bRFS whereas their incorporation along with current clinico- pathological prognostic markers did not changed the risk prediction for the high risk subgroup (LR=0.49). Interestingly, the presence of the UGT2B17 gene deletion is enriched in high risk patients, OR 3.83 (95% CI [2.31 1 ; 6.373]; p=2X10^"7), which is not the case for UGT2B28, OR 0.968 (95% CI [0.67; 1 .398]; p=0.863). Thus, the presence of UGT2B17 deletions would be associated with adverse clinical features. No other significant interaction was noted between gene markers, PSA, Gleason score and TNM staging (Table 8). DISCUSSION

[0069] We assessed the prognostic value of common genetic variants of five major sex- steroid metabolizing UGT isoforms in clinically localized and locally advanced prostate cancer patients following prostatectomy. We analyzed the samples of 1 161 patients with localized and locally advanced prostate cancer from four independent cohorts, for which prostatectomy was the sole initial therapeutic intervention. To our knowledge, there are no previous reports studying this inherited variation in UGT2B genes for prostate cancer recurrence. Using this data set, we observed a positive correlation between individuals with one or two deletion alleles for UGT2B17 and UGT2B28 and bRFS. However, no significant association between selected SNPs in other UGT genes and bRFS was observed in our study. Studying genes involved in the end of the androgenic signal is biologically relevant in the initial phase of disease development because prostate cancer cells are hormone responsive at this stage, and hormonal manipulation is a cornerstone of disease management. [0070] Our findings demonstrate that the presence of at least one deleted allele of the sex-steroid metabolizing gene UGT2B28, or the co-occurrence of two deletions in the UGT2B17 and UGT2B28 genes is predictive of early PSA failure after prostatectomy in both Asians and Caucasians. Recently, CNV in UGT2B17 was shown to be associated with osteoporosis in Asians,⁴² linking CNV for the first time with a complex metabolic disease. Of all genetic process involved in prostate cancer progression, CNV is certainly one of the most drastic molecular events. However, to date, very limited data have looked at the association between CNV and cancer risk and more importantly, none have been linked to cancer progression. CNV of UGT2B17 was shown to be associated with prostate cancer risk in some studies but not in others. ⁸'^26,27,29 However, none of these studies looked simultaneously at the chromosomal neighbors UGT2B15 and/or UGT2B28, which may contribute to the discrepancies observed.

[0071] Incorporation of CNV in UGT genes and other relevant genetic variants in a nomogram will ultimately provide a relevant clinical translation that could help guide treatment decisions. Indeed, we also revealed that the incorporation of UGT CNV status to the well recognized d'Amico risk classification system, improved stratification of low and intermediate risk patients. [0072] This data thus implicates CNV in sex-steroid metabolizing genes as biomarkers for prostate cancer recurrence, and provides support for the significance of sex-steroids in the pathogenesis and recurrence of this prostate cancer.

Example 2 - Genotyping of SRD5A genes polymorphisms Methods

Clinical Data and Outcome Collections

[0073] A second study was mostly composed of Caucasians, and included 526 men who underwent RP at I'Hotel-Dieu de Quebec Hospital (QC, Canada) between February 1999 and December 2002. Each participant provided written consent before surgery for the analysis of their genome and the research protocol was approved by the research ethical committee at the Centre Hospitalier Universitaire de Quebec (CHUQ, QC, Canada). All patients were followed postoperatively with serial PSA measurements and detailed clinical information was available.

DNA Isolation and Genetic Analysis

[0074] Polymorphisms in SRD5A genes were chosen according to one or more of the following criteria: i) to be likely functional (with supportive data in the literature), ii) to have previously been associated with PCa risk, aggressiveness, age at onset, BCR or ADT efficiency, and iii) to explain most of the haplotype diversity in the CEU (Utah residents with Northern and Western European Ancestry) Hapmap population. For both SRD5A genes, a region covering all the exons, introns and 5 kb of the 5' and 3' sections of the genes was screened using a haplotype tagging SNPs (htSNPs) strategy to maximize coverage, using HapMap Phase 2 ⁰, and data from the CEU unrelated subjects based on a r2 >0.80 and a minimum minor allele frequency >0.05. Determination of UGT2B genetic deletion status has been described elsewhere ³¹. [0075] Peripheral blood was collected on the morning of a preoperative clinic visit and kept frozen at -80°C until analysis. Genomic DNA was purified using the QIAamp DNA Blood Mini Kit (Qiagen Inc., Mississauga, On, Canada) and stored at -20°C. For htSNPs in SRD5A genes, PCR amplifications were performed using Sequenom iPLEX matrix- assisted laser desorption/ionization-time-of-flight mass spectrometry. For oligos sequence, see Table 12. Negative controls were present for every run of analyses and quality controls (random replicates of known genotypes) were successfully performed in 5% of the study cohort.

Statistical Analysis

[0076] Based on our sample size, we were able to detect a hazard ratio of 1.50, for a minor allele with frequency of 5%, with over 80% statistical power. Allelic frequencies and Hardy-Weinberg equilibrium were computed with PLINK (version 1.07) ⁴³. To analyze their association with BCR, each htSNP was considered using 3 models since the function of most htSNPs remains unknown. The genomic model considered the htSNP as a categorical variable with a common allele homozygote, heterozygote and a minor allele homozygote. The dominant model considered the htSNP with only 2 categories: one with a common allele homozygote and one with at least one minor allele. Finally, the recessive model also considered the htSNP with only 2 categories: one with at least one common allele and one minor allele homozygote. Cox regression was performed on each htSNP considering the 3 above mentioned models with adjustment for clinicopathological variables: PSA level at diagnosis, age at diagnosis, smoking status, pathological Gleason grade, pathological stage and neoadjuvant ADT. All co-variables were treated as categorical, and for PSA level, Gleason scores and stage, they were used as described by the D'Amico risk classification. The censoring variable was BCR, which was defined as 1 ) two consecutive PSA values > 0.3 pg/L, 2) one PSA value > 0.3 pg/L followed by ADT, and 3) a last-recorded PSA value > 0.3 pg/L after prostatectomy.

[0077] Kaplan-Meier analyses were also processed for every htSNP (log-rank), but only results for htSNPs significantly associated with BCR in Cox regression multivariate analysis are shown. These statistical analyses were performed using PASW statistics 17 (SPSS Inc., Chicago, IL) and R version 2.10.0. Results

[0078] All 526 patients had initially RP as curative intent enabling a precise pathological evaluation. Clinical and pathological characteristics of the study cohort are shown in Table 9. The median follow-up time of the cohort is 7.4 years (range: 0.5 to 10.2 years). The cohort had mainly organ-confined and locally advanced tumors, as PCa cases were composed of pT2 (60%) and pT3 (37%) pathological tumor stages (Table 9). Overall, 130 cancer cases experienced BCR (25%), with a median time to relapse of 2.1 years. [0079] Risk of recurrence associated with known clinical and pathological prognostic variables are shown in Figure 7. The risk of recurrence increased with higher PSA values at diagnosis with a risk of relapse of HR= 1.5 (P=0.081 ) and HR=2.1 (P=0.003) for PSA values of >10-20 ng/mL and >20 ng/mL, respectively. Gleason scores of 7 and≥ 8 were also positively associated with relapse with HR of 2.6 (P=0.002) and 5.4 (P=0.0000003), respectively.

[0080] A total of 19 htSNPs distributed across the two SRD5A genes were studied herein, of which 2 were functional coding SNPs. htSNPs were selected with a strategy to maximize gene coverage and to reflect adequately the Caucasian haplotype genetic diversity. The htSNPs strategy allowed us to study 109 genetic variations in both genes (Table 13).

[0081] After analyses with a Cox regression multivariate model, adjusted for all clinical and pathological factors known to affect BCR, 7 htSNPs, 2 in SRD5A1 and 5 in SRD5A2, were positively associated (P<0.05) with the risk of relapse. Their relative frequencies in cancer patients with and without relapse, and the corresponding hazard ratios (95% CI) are displayed in Figure 7B and Table 10. Genetic linkage between the 19 htSNPs tested in both SRD5A genes is also represented.

[0082] Among 7 htSNPs tested in SRD5A1 , the rs518673 showed a P=0.008 by log-rank test for recurrence-free survival while the rs166050 was almost significant with a (log-rank test P=0.051 ; Figure 8). The SRD5A1 rs166050 gene polymorphism was associated with an increased recurrence risk of HR=1 .89 (95% CI, 1 .03-3.45; P=0.039), while the rs518673 in SRD5A1 was associated with a decreased recurrence risk (HR=0.63, 95% CI, 0.44-0.91 ; P=0.014; Table 10).

[0083] Of the 5 SRD5A2 htSNPs associated with the risk of BCR, a significant association was observed with the nonsynonymous SNP V89L (rs523349) while no association was seen with the other known coding variation A49T (rs9282858) (HR=0.81 , 95% CI, 0.36- 1.85; P=0.62) (Table 10). Additional variations in SRD5A2 were also associated with increased risk of recurrence; rs2208532, rs676033 and rs4952197 with HR of 1 .65, 1.77 and 2.05, respectively (Figure 7B and Table 10). In contrast, the SRD5A2 htSNP rs 12470143 was associated with a reduced risk of recurrence; HR of 0.64 (95% CI, 0.44- 0.93; P=0.019). Kaplan-Meier and log-rank P values are illustrated in Figure 8B. [0084] By combining protective alleles in SRD5A1 (rs518673 T) and SRD5A2

(rs12470143 A), the effect was shown to be greater in Kaplan-Meier analysis with the maximum protection conferred by 3 or 4 alleles (Figure 8C) and remained an independent predictor of recurrence in Cox proportional hazards analysis (HR=0.34, 0.18-0.64;

P=0.0009; Table 11 ). We also discovered a significant allele-dose-dependent relationship with recurrence in which each additional allele diminishing by 26% the risk of recurrence of BCR (P=0.004).

[0085] Here, we outlined a significant association of BCR with multiple genetic

polymorphisms in both SRD5A genes in a cohort of 526 PCa cases. Remarkably, of the 19 htSNPs tested, 7 inherited variations (37%) were shown to affect the risk of recurrence after RP either by conferring protection or an elevated risk, independently of known clinical and pathological predictors of prostate cancer recurrence. Our study is the first to show strong positive associations of multiple genetically unlinked SRD5A2 variations with BCR and to argue for a significant role of SRD5A1 in the risk of recurrence after RP.

These findings thus define that genetic variations in both 5a-reductase type 1 and type 2 pathways play a critical role in prostate cancer recurrence after RP while the effect was magnified with an increasing number of SRD5A adverse genotypes. Furthermore, a protective effect by two unlinked SRD5A genetic variants, rs518673T and rs12470143A, on recurrence was revealed. Reduced occurrence of BCR was shown to be maximal in patients with 3 to 4 of these protective alleles in both SRD5A genes.

[0086] One of the best characterized SNP in the SRD5A2 gene, the functional V89L polymorphism, is associated with a decreased enzymatic activity.^44,45 This polymorphism has been associated with prostate cancer risk in some studies^32,34 agressiveness^{32 33}, and with conflicting results with biochemical recurrence^{12 13}. One important finding of our study is a significant association between the V89L (rs523349) polymorphism and the risk of BCR after prostatectomy (2.1 -fold risk of recurrence for homozygotes rs523349C). This result is in agreement with Shibata and colleagues ²⁷ and with a recent large study that associated this SNP with an aggressive form of PCa³³. However, for the non-synonymous SRD5A2 A49T variant (rs9282858), a functional SNP associated in some studies with the risk of PCa⁴⁶, no association was evidenced with recurrence-free survival after RP indicating that this SNP would have no obvious role in PCa recurrence. This result is consistent with a meta-analysis investigating this SNP⁴⁶. Example 3 - Co-occurrence of polymorphism in the SRD5A and copy number variations UGT2B genes

[0087] As presented herein before, patients with two or more deleted copies of the UGT2B17 and/or UGT2B28 genes have an increased risk of recurrence (HR of 2.12 (95% CI [1 .34; 3.37]). Again, the presence of a SRD5A risk alleles combined with deletions in UGT2B genes further increased this risk (Figure 9 and Table 14). For example, when combining the group of patients carrying risk alleles in UGT2B and SRD5A2 rs523349C, those individuals had more than 70% BCR rates compared to 25% for the other groups (Log-rank P=0.000007; Figure 9). The HR values rose from 2.12 (95% CI; 1.21 -3.75; P=0.009) for individuals homozygous SRD5A2 rs523349C to 4.97 (95% CI, 2.38 - 10.36; P=0.00002; Figure 9) for patients with deletions in the UGT2B genes. Similarly, the presence of a rs2880532 SRD5A2 risk allele combined with deleted copies of UGT2B are associated with more than 50% BCR, compared to less than 25% for the other groups (Log-rank P=0002; Figure 9). Patients homozygous for rs2208532G and rs676033A had HR values of 1 .65 and 1.77 respectively; compared to risks of 2.85 and 3.85 when combined with UGT2B risk alleles (Figure 9).

[0088] In patients with unfavourable UGT2B genotypes (e.g. deleted copies) but carrying 3-4 protective SRD5A alleles, BCR-free recurrence reached 80.8% compared to 37.5% for those patients with no protective allele (LR: 0.001 ; Table 11 and Figure 10).

Comparable BCR-free survival rates (LR=0.17) were observed in patients with no deleted copies of UGT2B presented, regardless of the presence of SRD5A protective alleles.

[0089] As a result, we observed an additive effect of UGT2B CNVs with htSNPs in SRD5A genes. Indeed, as presented hereinbefore, we demonstrated that deletions of both UGT2B17 and UGT2B28 are associated with risk of recurrence of PCa after RP. As both SRD5A and UGT enzymes are the rate limiting steps in T and DHT

biotransformation, we confirmed that genetic alterations in both pathways are additive and strikingly modify the risk of biochemical relapse after RP.

[0090] Of note, this data set included a large sample size combined with relevant information on clinical and pathological covariates as well as a significant median follow- up time of 7.4 years. However the limited number of some clinically relevant events such as metastasis, androgen-deprivation resistance, or death related to the localized features of the tumors, prevented us from addressing the association between inherited variations in candidate genes and risk for these events.

[0091] This data clearly demonstrated for the first time that multiple genetic markers in SRD5A and UGT2B genes contribute to biochemical recurrence risk after RP. In localized disease clinical settings, these markers are independent of current predictors of recurrence such as Gleason score and PSA level and predict risk better than the pathological stage. These findings ultimately help refine our ability to identify individuals at low or high risk of cancer relapse after RP, beyond known prognostic variables, and for whom a more personalized approach might optimize outcome, especially in the context of PCa chemoprevention using of 5a-reductase inhibitors therapy. Indeed, the inclusion of such markers is critical in predicting clinical outcome and may lead to informed decisions such as for endocrine treatment with major implications on the patient's recurrence-free survival and quality of life.

References

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Claims

1. An in-vitro method for predicting risk for prostate cancer in a human subject, said method comprising the steps of:

a) obtaining a nucleic acid from sample from said human subject; and b) determining the individual's UGT2B genetic variation status;

whereby the identification of at least one genetic variation in said UGT2B gene in said subject's nucleic acid is an indication that said subject has an increased risk of developing prostate cancer or progression thereof.

2. The method of claim 1 , wherein said UGT2B gene is selected from the group consisting of: UGT2B17 and UGT2B28.

3. The method of claim 2, wherein said genetic variation in UGT2B17 is one or two deletions,

4. The method of claim 2 or 3, wherein said genetic variation in UGT2B28 is one or two deletions.

5. The method of claim 2, 3 or 4, wherein said identification of genetic variation in said UGT2B17 or UGT2B28 comprises identifying at least one copy number variation (CNV) in said gene, said CNV being selected from the group consisting of: rs3100645; and rs1 1249532, and associated reference sequences thereof.

6. The method of claim 2, 3, 4 or 5, comprising at least one genetic variation in both UGT2B17 and UGT2B28 genes.

7. The method of claim 1 to 6, further comprising at least one genetic variation in SRD5A1 or SRD5A2 gene or both; whereby the co-occurence of at least one genetic variation in UGT and SRD5A1 or SRD5A2 in said subject's nucleic acid is an indication that said subject has a further increased likelihood that the prostate cancer will recur.

8. The method of claim 7, wherein said identification of genetic variation in said SRD5A1 or SRD5A2 comprises identifying at least one single nucleotide polymorphism (SNP) in said nucleic acids, said SNP being selected from the group consisting of: rs166050; rs2208532; rs523349; rs4952197; rs12470143; rs518673 and rs676033, and associated reference sequences thereof.

9. The method of claim 1 , wherein in step a) said nucleic acid is DNA or RNA.

10. The method of claim 9, wherein said genetic variation of DNA or RNA is detected with the use of a nucleic acid probe.

11. The method of claim 10, wherein said DNA is amplified by PCR prior to incubation with the probe.

12. The method of any one of claims 1 to 10, wherein said genetic variation of DNA or RNA is detected with the use of a methodology selected from the group consisting of: direct sequencing, pyrosequencing, massively parallel sequencing, high-throughput sequencing, high performance liquid chromatography (HPLC) fragment analysis, mass spectrometry, branch DNA and capillary electrophoresis.

13. The method of claim 12, wherein said methodology is carried out on DNA or RNA with prior amplification.

14. The method of claim 13, wherein said genetic variation in said DNA is detected with the use of a specific primer and a specific probe.

15. The method of claim 14, wherein the primer is selected from the group consisting of: SEQ ID Nos. 1 , 2, 7 or 8, and the probe hybridizes with a sequence selected from rs3100645 and/or rs1 1249532m, and associated reference sequences thereof.

16. The method of claim 1 , wherein the subject's nucleic acid-containing sample is a tumor or a non-tumor sample.

17. The method of claim 16, wherein the subject's non-tumor sample is selected from the group consisting of: tissue or biological fluid.

18. The method of claim 17, wherein said tissue sample is selected from the group consisting of: skin, lymph node, hair and buccal smear.

19. The method of claim 17, wherein said biological fluid sample is selected from the group consisting of: sputum, saliva, blood, serum, urine, semen and plasma.

20. The method of claim 17, wherein said tumor sample is obtained from a biopsy.

21. A method for adapting a course of treatment of prostate cancer in a human subject, comprising the steps of:

a) providing a prognosis or predicting the likelihood of a human subject to develop prostate cancer recurrence in accordance with any one of claims 1 to 20; and

b) adapting a course of treatment according to whether said subject has an increased likelihood that the cancer will recur.

22. The method according to claim 21 , wherein, when the likelihood of recurrence is increased, the subject is prescribed at least one of: hormone therapy, chemotherapy, surgery, radiotherapy, brachytherapy or active surveillance (watchful waiting).

23. The method according to claim 20 or 21 , wherein steps a) and b) are performed after the subject has undergone radical prostatectomy.

24. The method according to any one of claims 21 to 23, wherein, when the likelihood of recurrence is increased, the subject is prescribed UGT2B inducer therapy.

25. The method according to any one of claims 21 to 23, wherein, when the likelihood of recurrence is increased, the subject is prescribed 5a-reductase inhibitors therapy.

26. A kit for predicting the likelihood of a human subject to develop prostate cancer recurrence by detecting a genetic variation according to any one of claims 1 to 8; said kit comprising reagents for determining the individual's genetic variations (or haplotypes) in UGT2B gene.

27. The kit of claim 26, comprising a reagent for detecting a reference sequence selected from the group consisting of: rs3100645; and rs1 1249532; and associated sequences thereof.

28. The kit of claim 25 or 26, comprising a PCR primer-probe set, wherein the primer is selected from the group consisting of: SEQ ID Nos. 1 , 2, 7 or 8 and the probe hybridizes with a sequence selected from rs3100645 and/or rs1 1249532, and associated reference sequences thereof.

29. The kit of claim 28, further comprising reagents for determining the individual's genetic variations (or haplotypes) in SRD5A1 or SRD5A2 gene.

Particularly, the kit further comprises reagents for detecting a SNP in a reference sequence selected from the group consisting of: rs518673; rs166050; rs12470143;

rs2208532; rs523349; rs4952197 and rs676033 or their associated SNPs.

30. The kit of claim 29, comprising PCR primer-probe set, wherein the primer is selected from the group consisting of: SEQ ID Nos. 12 to 50; and the probe is selected from the group consisting of: SEQ ID Nos. 51 to 69.

31. A method of predicting the response to hormonotherapy for treating prostate cancer in a subject, the method comprising carrying the steps according to any one of claims 1 to 25; whereby the determination of at least one genetic variation in said UGT2B gene in said subject's nucleic acid is an indication that said treatment regimen is not efficacious for treating prostate cancer in the subject.

32. A method of predicting the response to 5-alpha inhibitor therapy for treating prostate cancer in a subject, the method comprising carrying the steps according to any one of claims 7 or 8; whereby the determination of at least one genetic variation in said SRD5A1 or SRD5A2 gene in said subject's nucleic acid is an indication that said treatment regimen is not efficacious for treating prostate cancer in the subject.

33. A biomarker for predicting the likelihood of prostate cancer progression or recurrence in a human subject, comprising a genetic variation in UGT2B17 or UGT2B28 gene of said human subject.

34. A genetic variation in UGT2B17 or UGT2B28 gene of a human subject for use as a biomarker for predicting the likelihood of prostate cancer progression or recurrence in said human subject.