Narasimhan Ragavan

SOX9 Elevation in the Prostate Promotes Proliferation and Cooperates with PTEN Loss to Drive Tumor Formation

Dysregulation of tissue development pathways can contribute to cancer initiation and progression. In murine embryonic prostate epithelia, the transcription factor SOX9 is required for proper prostate development. In this study, we examined a role for SOX9 in prostate cancer in mouse and human. In Pten and Nkx3.1 mutant mice, cells with increased levels of SOX9 appeared within prostate epithelia at early stages of neoplasia, and higher expression correlated with progression at all stages of disease. In transgenic mice, SOX9 overexpression in prostate epithelia increased cell proliferation without inducing hyperplasia. In transgenic mice that were also heterozygous for mutant Pten, SOX9 overexpression quickened the induction of high-grade prostate intraepithelial neoplasia. In contrast, Sox9 attenuation led to a decrease proliferating prostate epithelia cells in normal and homozygous Pten mutant mice with prostate neoplasia. Analysis of a cohort of 880 human prostate cancer samples showed that SOX9 expression was associated with increasing Gleason grades and higher Ki67 staining. Our findings identify SOX9 as part of a developmental pathway that is reactivated in prostate neoplasia where it promotes tumor cell proliferation.

GENE EXPRESSION PROFILING OF THE HUMAN PROSTATE ZONES

To investigate differences in gene expression in different zones of the prostate by microarray analyses, to better understand why aggressive tumours predominantly occur in the peripheral zone (PZ), whereas benign prostatic hyperplasia (BPH) occurs almost exclusively in the transition zone (TZ).

Quantification of phase I / II metabolizing enzyme gene expression and polycyclic aromatic hydrocarbon-DNA adduct levels in human prostate

Studies of migrant populations suggest that dietary and/or environmental factors play a crucial role in the etiology of prostatic adenocarcinoma (CaP). The human prostate consists of the peripheral zone (PZ), transition zone (TZ), and central zone (CZ); CaP occurs most often in the PZ.

Constitutive expression of bioactivating enzymes in normal human prostate suggests a capability to activate pro‐carcinogens to DNA‐damaging metabolites

The constitutive bioactivating capacity of human prostate may play a role in determining risk of adenocarcinoma developing in this tissue. Expression of candidate enzymes that convert exogenous and/or endogenous agents into reactive DNA‐damaging species would suggest the potential to generate initiating events in prostate cancer (CaP).

Differential gene expression in the peripheral zone compared to the transition zone of the human prostate gland.

Gene expression profiles may lend insight into whether prostate adenocarcinoma (CaP) predominantly occurs in the peripheral zone (PZ) compared to the transition zone (TZ). From human prostates, tissue sets consisting of PZ and TZ were isolated to investigate whether there is a differential level of gene expression between these two regions of this gland. Gene expression profiling using Affymetrix Human Genome U133 plus 2.0 arrays coupled with quantitative real-time reverse transcriptase-PCR was employed. Genes associated with neurogenesis, signal transduction, embryo implantation and cell adhesion were found to be expressed at a higher level in the PZ. Those overexpressed in the TZ were associated with neurogenesis development, signal transduction, cell motility and development. Whether such differential gene expression profiles may identify molecular mechanisms responsible for susceptibility to CaP remains to be ascertained.

Quantified gene expression levels for phase I/II metabolizing enzyme and estrogen receptor levels in benign prostate from cohorts designated as high-risk (UK) versus low-risk (India) for adenocarcinoma at this organ site: a preliminary study.

Risk of clinically significant prostate adenocarcinoma (CaP) varies worldwide, although there is a uniform prevalence of latent disease. A hormone-responsive tissue, the prostate possesses the metabolizing capacity to biotransform a variety of environmental procarcinogens or endogenous hormones. Whether such metabolizing capacity or estrogen receptor (ER) status underlies these demographic differences in susceptibility to CaP remains unclear. With appropriate ethical permission, verified-benign tissues were obtained following transurethral resection of the prostate from a high-risk region (n = 12 UK-resident Caucasians) and a typically low-risk region (n = 14 India-resident Asians). Quantitative gene expression analysis was employed for cytochrome P450 (CYP)1B1, N-acetyltransferase (NAT)1, NAT2, catechol-O-methyl transferase (COMT), sulfotransferase (SULT)1A1, ERalpha, ERbeta and aromatase (CYP19A1). To quantify the presence or absence of CYP1B1, ERalpha or ERbeta, and to identify their in situ localization, immunohistochemistry was carried out. The two cohorts had reasonably well-matched serum levels of prostate-specific antigen or hormones. Expression levels for the candidate genes investigated were similar. However, clear differences in protein levels for CYP1B1 and ERbeta were noted. Staining for CYP1B1 tended to be nuclear-associated in the basal glandular epithelial cells, and in UK-resident Caucasian tissues was present at a higher (P = 0.006) level compared with that from India-resident Asians. In contrast, a higher level of positive ERbeta staining was noted in prostates from India-resident Asians. These study findings point to differences in metabolizing capacity and ER status in benign prostate tissues that might modulate susceptibility to the emergence of clinically significant CaP in demographically distinct populations.

Risk of prostate cancer after detection of isolated high-grade prostatic intraepithelial neoplasia (HGPIN) on extended core needle biopsy: a UK hospital experience

BackgroundHigh-grade prostatic intraepithelial neoplasia (HGPIN) is a precursor lesion to prostate cancer (CaP). UK-based studies examining the occurrence of isolated HGPIN and subsequent risk of CaP are lacking. Our aim was to assess the occurrence of HGPIN in a regional UK population and to determine whether in a retrievable cohort of such patients that had repeat extended core biopsies, there was an elevated risk of CaP.MethodsA retrospective analysis of the pathology database was conducted at our institution (Lancashire Teaching Hospitals NHS Foundation Trust) for prostate biopsies recorded between January 2001 and December 2005 (all extended core biopsies). Those patients with isolated HGPIN on 1st set of biopsies were identified and, their clinical characteristics and pathological findings from subsequent biopsies (if any) were determined. The risk of CaP on subsequent biopsies based on presenting baseline PSA was stratified.ResultsOf 2,192 biopsied patients, there were 88 cases of isolated HGPIN of which 67 patients underwent one or more repeat biopsies. In this repeat-biopsy group, 28 CaP diagnoses were made. Age at first biopsy (P < 0.001), higher mean baseline prostate-specific antigen (PSA) (P < 0.005) and higher mean change in PSA (P < 0.05) were predictive of CaP detection on repeat biopsies. PSA ranges and their associated predictive values for cancer were: 0 to 5 ng/ml – 11%; 5 to 10 ng/ml – 34%; 10 to 20 ng/ml – 50%; and > 20 ng/ml – 87.5%.ConclusionBased on our results, we recommend delaying the 1st repeat biopsy at low PSA range but to have a shorter interval to repeat biopsies at intermediate and higher PSA ranges.

Expression of hormone-/carcinogen-metabolising enzymes in the prostate : clues into peripheral-zone susceptability?

INTRODUCTION & OBJECTIVES: The peripheral zone (PZ) of the prostate presents with a higher occurrence of adenocarcinoma (CaP) compared to transition zone (TZ). Environmental procarcinogens and endogenous hormones implicated in the aetiology, often requires bio-activation to DNA-binding species (that form DNA-carcinogen adducts) which are done by phase I cytochrome P-450 (CYP) isoenzymes (CYP1A1, CYP1A2 and CYP1B1) and, the phase II N-acetyl transferases (NAT1 and NAT2) and catechol-O-methyl transferase (COMT). The objective of this study is to assess intra- (PZ vs. TZ) and inter-individual variations in the gene expression of phase I and II enzymes using quantitative real-time RT-PCR in CaP-free tissues. MATERIAL & METHODS: With ethical approval, prostate tissue sets (PZ and TZ) (n=27) were obtained from patients (inclusion criteria - low PSA (<20 mg/l serum) and/or low volume disease ≤two/eight core biopsies positive for CaP)) undergoing radical prostatectomy, isolated from a lobe preoperatively identified as negative for CaP. Real-time RT-PCR was employed to quantitatively examine CYP1A1, CYP1A2, CYP1B1, NAT1, NAT2 and COMT. Immunohistochemistry (with polyclonal anti-CYP1B1 antibody) was employed to assess CYP1B1 protein and location in the prostate. In all cases, retrospective analysis (after HE some nuclear staining was also observed in the stroma. In CaP tissue, sheaths of cells exhibiting both nuclear staining and cytoplasmic staining were observed. CONCLUSIONS: Our study demonstrates the expression in the prostate of phase I and II enzymes. And CYP1B1 expression is particularly high in peripheral zone. Its role as a target either for chemoprevention/treatment strategies remains to be investigated.

Re: Urs E. Studer, Laurence Collette, Peter Whelan, et al. Using PSA to Guide Timing of Androgen Deprivation in Patients with T0–4 N0–2 M0 Prostate Cancer not Suitable for Local Curative Treatment (EORTC 30891). Eur Urol 2008;53:941–9

We read the article by Studer et al [1] with great interest. The timing for androgen deprivation therapy (ADT) in prostate cancer (PCa) is crucial and remains largely unclear. In this study, the authors set out to assess whether serum levels of prostate-specific antigen (PSA) might be used as a guide for when to best initiate ADT. While this question is of significant interest, in light of their findings, we feel that the authors have made certain bold assertions. The statement, ‘‘Our analyses suggest that patients with a baseline PSA >50 ng/ml are likely to die of PCa and therefore are good candidates for immediate ADT,’’ is slightly misleading. Additionally, when assessing the paper and its data, one might come to the following conclusions:

CYP1B1 expression is higher in the peripheral zone compared to the transition zone : a difference underlying zonal susceptibility to prostate adenocarcinoma?

Introduction The prostate is a composite organ divided into zones. Prostate adenocarcinoma (CaP) mostly occurs in the peripheral zone. Cytochrome P-450 (CYP) isoenzymes are constitutively expressed and inducible enzymes, many of which (e.g. CYP1A1, CYP1A2 and CYP1B1 isoforms) are involved in hormone and carcinogen hydroxylation. Aim To assess variations in CYP expression in the prostate on an intra-(peripheral zone Vs. transition zone) and inter-individual basis. Materials and Methods Human prostates (n=24) were obtained, with ethical approval, from radical retropubic prostatectomies. Study participants exhibited low PSA (< 20 μg/l serum) and low volume of disease (< two/eight core biopsies positive for CaP). Following resection, tissue sets consisting of peripheral zone and transition zone were isolated from a lobe pre-operatively identified as negative for CaP. A histopathologist always examined adjacent tissue. Real-time RT-PCR was employed to quantitatively examine CYP1A1, CYP1A2 and CYP1B1. Results CYP1A1 mRNA transcripts were detected in either or both zones of twenty tissue sets (in seven cases, in both zones) and were undetectable in four others. In eleven tissue sets, higher levels of CYP1A1 expression were observed in the peripheral zone compared to the transition zone (maximum six-fold difference) whereas in nine other tissue sets this relationship (maximum 2.5- fold difference) was reversed. CYP1A2, although detectable in twelve tissue sets, was not quantifiably expressed. CYP1B1 expression was detected in both zones of all tissue sets examined. Inter-individual variation in CYP1B1 expression levels in peripheral zone (five-fold differences) and transition zone (tenfold differences) were noted. In sixteen out of seventeen tissue sets found to be cancer free, CYP1B1 expression was found to be two- to fifty-fold higher in the peripheral zone compared to the transition zone; in the remaining tissue set an equal level of gene expression was detected in both zones. In the tissue sets containing CaP, CYP1B1 expression was higher in the cancerous zone (be it peripheral or transition) in five of six cases; in another tissue set containing PIN, an equal level of gene expression was again detected in both zones. Immunohistochemistry clearly demonstrated a nuclear staining pattern for CYP1B1 in epithelial and stromal cells of both zones; the staining density of this protein was markedly elevated in cancerous tissue. Discussion CYP1B1 preferentially catalyses the 4-hydroxylation of 17β- oestradiol, metabolically activates exogenous pro-carcinogens and inactivates anticancer agents. Future studies will investigate whether CYP1B1 may be employed as a target either for chemoprevention strategies or treatment of clinically invasive disease.British Prostate Group Prostate Cancer and Prostatic Diseases Introduction: Prostate cancer is considered a tissue diagnosis commonly based on biopsy cores. Standard prostate biopsy has a reported minor complication rate of 60-79%, major complication rate of 0.44.4% and the need for hospitalisation in 0.4-3.4% of men (Norberg Eur.Radiol. 1996; 6: 457, Aus Br.J.Urol. 1996; 77: 851, Rietbergen Urology 1997; 49: 875, Rodriguez J Urol. 1998; 160: 2115-20) and infection risk increases with age (Lindert J Urol. 2000; 164: 76). This study examines whether prostatic biopsies are necessary in all men aged 80 or above. Methods: The pre-biopsy PSA, the DRE, the biopsy findings and staging bone-scan results of all men aged ≥80 years who underwent prostatic biopsies between 2000-2003 were reviewed. All biopsy samples had been examined in one of three histopathology units and thirty-three consultant urologists contributed. Results: 211 men ≥ 80 years were identified, of whom 163(77%) had biopsy proven prostate cancer. 100% of 29 men with PSA ≥100, 98% of 47 with PSA ≥50, 97% of 77 with PSA ≥30 and 92% of 102 with PSA ≥20ng/ml had biopsy cores containing cancer. 63% of men with PSA <20ng/ml had cancer on biopsy. In men with cancer and a PSA ≥30ng/ml, 92% had Gleason grade ≥7and 93% were treated with antiandrogen therapy and /or pelvic radiotherapy. In all men with cancer the DRE was abnormal in 91%, the mean number of positive cores was 59% and the bonescan was positive in 18%. The DRE was abnormal in 77% of men with benign biopsies. Conclusions In men ≥ 80 years with a PSA ≥30ng/ml, at least 97% had prostate cancer, over 90% of these men had high-grade disease and nearly all men with cancer received active treatment. The value of prostatic biopsy in this age group, with PSA ≥30ng/ml, is questionable. Sensitive and non-invasive diagnosis of Prostate Cancer using E2F3 quantitative RT-PCR C. P. Pipinikas, S. B. Nair , K. I. Konstantinou, R. S. Kirby, N. D. Carter and C. D. Fenske St George’s Hospital and Medical School, London SW17, Department of Epidemiology and Public Health, Imperial College, Faculty of Medicine, London W2 1PG Introduction Reverse transcriptase-polymerase chain reaction (RT-PCR) is a sensitive molecular technique capable of detecting prostate cells in peripheral circulation. Quantitative RT-PCR (qRT-PCR) is an expansion of this technique, used to look at levels of gene expression. Potential markers for prostate cancer (CaP) have been assessed using qRT-PCR with the aim of more accurate diagnosis. E2F3 gene, a member of the E2F family of cell cycle regulatory transcription factors (Oeggerli et al. Oncogene 2004; 23:5616-23) has been shown to be overexpressed in CaP, controlling proliferation rates and dictating CaP aggressiveness Furthermore E2F3 directly modulates the expression of the EZH2 gene, which also controls cellular proliferation and is upregulated in CaP (Foster et al. Oncogene 2004; 23: 5871-9). Both genes, therefore, are potentially excellent candidate markers for CaP diagnosis. Using relative quantitative RT-PCR (qRTPCR), E2F3 and EZH2 expression was analysed as potentially accurate markers for CaP. Methods Total RNA was extracted in quadruplet from blood taken from 110 patients. cDNA synthesis was carried out, followed by qRTPCR using E2F3 and EZH2 gene specific primers and the LightCyclerTM (Roche). Results Relative qRT-PCR for E2F3 was carried out on four distinct patient groups, based on detailed clinicopathological information. E2F3 expression showed highly significant differences between all patient groups (P<0.001): a 39-fold increase was found in the localised prostate cancer group (LocCaP: n=51;mean= 4.67) compared to benign prostatic hyperplasia group (BPH: n=8; mean=0.12). Levels in the metastatic prostate cancer group (MetCaP: n=23; mean =1.64) were lower than the LocCaP group, but still 14-fold higher than the BPH group levels. Of particular interest was the radical prostatectomy group (RP: n=18, mean = 4.08), which showed levels of E2F3 expression similar to those of the LocCaP group. This would indicate the presence of tumour cells in peripheral circulation, suggesting undetected micrometastases. No E2F3 expression was detected in male control samples (n=10). Preliminary results for EZH2 showed a similar expression profile to that of E2F3, with significant upregulation in CaP patients. Discussion The expression of E2F3 has been shown to be highly upregulated in prostate cancer. Results show that it can be used as a marker in accurate diagnosis of CaP using the non-invasive, sensitive technique of qRT-PCR. E2F3 has been shown to directly modulate the expression of cellular proliferation genes, including EZH2, and is linked to pRB, a cell cycle protein (Huang et al Nat Genet 2003; 34:226230). It is therefore possible that the pRB-E2F3-EZH2 axis represents an underlying molecular mechanism involved in the development of CaP. This offers the potential of specific treatment based on patient’s gene expression profile. Prostate Cancer The MALE Perspective Michael G. Clarke, JRM Wilson, JD Graham, RP MacDonagh Taunton & Somerset Hospital, Taunton Introduction It is essential when making judgements regarding the management of prostate cancer, that clinicians utilise both patient choice and life expectancy, in addition to the available clinical parameters. However estimations of life expectancy, based on comorbidity, are often inaccurate and inconsistent. Whilst validated co-morbidity scores exist, these are often disease-specific and cumbersome to use. We therefore aimed to develop a computer software program that would enable clinicians (or other members of the multidisciplinary team) to accurately calculate an individual patient’s life expectancy on the basis of their comorbid factors, with the potential for use in the clinical and MDT setting. Materials & methods In collaboration with a professional actuary, actuarial tables were used in association with the ‘numerical rating system’ to derive mortality ratios. These evidence-based actuarial mortality ratios, which are used by the insurance industry and are continually updated in line with the available medical literature, are calculated on the basis of a patient’s age, smoking habit and comorbidity. They can therefore provide an accurate method of life expectancy assessment in patients with multiple co-morbid factors. 17 medical conditions were chosen, since they 333 Prostate Cancer and Prostatic Diseases Abstracts British Prostate Group