Prostate Cancer and Prostatic Diseases (2003) 6, Suppl 1, S19–S28 & 2003 Nature Publishing Group All rights reserved 1365-7852/03 $25.00 www.nature.com/pcan Cytoreductive efficacy of goserelin and bicalutamide prior to radiotherapy

SEM Langley1, A Hendersen1 and R Laing1 1St Luke’s Cancer Centre, Royal Surrey County Hospital, Guildford, UK

Prostate Cancer and Prostatic Diseases (2003) 6, Suppl 1, S19–S28. doi:10.1038/sj.pcan.4500689

Introduction and objectives fall in the bicalutamide group and a 26% fall in the goserelin group. As initial prostate volume was not Androgen deprivation is commonly used to reduce equivalent in the three groups, a subgroup analysis prostate volume and hence avoid pubic arch interference was performed on patients who received active treat- during prostate brachytherapy. It has also become ment for 43 months who had initial prostate volume standard practice to give a short course of neoadjuvant o55 cm3. In this subgroup, a mean fall in prostate androgen suppression prior to radical external beam volume of 7% occurred in the bicalutamide group radiotherapy. It is thought that the efficacy of this compared with 21% in the goserelin group. In both the combined treatment is due to prostate volume reduction original and subgroup analyses, the cytoreductive that allows the treatment volume to receive a higher dose efficacy of goserelin was significantly greater than of radiation while sparing other tissues. We compare the bicalutamide (Po0.0001). cytoreductive efficacy of bicalutamide 150 mg or goser- elin with no hormonal manipulation in prostate cancer prior to brachytherapy. Conclusions Material and methods While our data suggest that LHRH analogues are Transrectal ultrasound volume estimations were per- superior in reducing the prostate volume when com- formed in clinic and during the brachytherapy planning pared to bicalutamide in patients prior to brachytherapy, scan. Between volume estimations patients received no this finding must be viewed with a degree of caution as hormonal treatment, bicalutamide 150 mg once daily or this was not a randomized study and is therefore open goserelin 3.6 mg every 28 days. to confounding by indication (where a difference is due to different populations studied and not treatment efficacy). Results If the beneficial effects of neoadjuvant hormone therapy in external beam radiotherapy are reliant upon Patients receiving no hormonal manipulation had a a reduction in prostate volume, then goserelin may also volume increase of 8% compared with an 8% volume be preferable to bicalutamide in this setting.

No Rx Bicalutamide Goserelin Bicalutamide Goserelin (all pts) (all pts) (o55 cm3 prostate, (o55 cm3 prostate, 43 m Rx) 43 m Rx)

N 34 66 31 55 19 Mean initial prostate volume (cm)3 (s.d.) 33.1 (6.6) 34.7 (11) 48.9 (16) 33.7 (10.7) 38 (11.3) Mean time between U/S in days (s.d.) 128 (78) 145 (64) 192 (83) 160 (54) 180 (60) Mean prostate volume change % (s.d.) +8 (16) À8 (15) À26 (14) À7 (15) À21 (11) P-value for paired t-test comparing 0.011 (33) o0.0001 (65) o0.0001 (30) 0.001 (54) o0.0001 (18) volume assessments (df) Abstracts S20 11Citrate-acetate PET in patients with prostate cancer

S Machtens1, E Fricke2, A Boerner3, C Stief1, M Hofmann3, W Knapp3, U Jonas1 1Department of Urology, Hannover Medical School, Germany; 2Department of Nuclear Medicine, Heart and Diabetes Center, Bad Oeynhausen, Germany; and 3Department of Nuclear Medicine, Hannover Medical School, Germany

Introduction and objectives Results Visualization of primary prostate cancer and its meta- The primary tumours were visualized in 20/24 (83%) stases has been a clinical problem, state-of-the-art patients using the 11C-acetate PET and in 10/15 (75%) methogs like CT, MRI and 18F-FDG PET nonwithstand- patients using the F-FDG PET. Based on the results of ing. The difficulties are even more pronounced in both scans, one patient was diagnosed with a lung cancer case of recurrence. While useful in the detection of as second primary. Average 18F-FDG uptake (SUV) was lymph-node and distant metastases the investigation of higher in distant metastases, whereas 11C-acetate uptake local recurrence with 18F-FDG positron emission tomo- (SUV) was higher in local recurrence and in local lymph- graphy (PET) is not considered helpful. The aim of node metastases. A closer relation between Gleason the study was to evaluate 11C-acetate in comparison to sum and PSA level on the one hand and 11C-acetate 18F-FDG PET in the detection of prostate cancer and its uptake was documented than for 18F-FDG uptake in metastases in patients treated with or without hormonal localized disease on the other hand. Either PETs ablation. demonstrated a reduced uptake in response to androgen ablation therapy.

Material and methods Conclusions A total of 25 patients (age: 54–82 y) were investigated These data deliver evidences that 11C-acetate PET might during follow-up of a primary prostate cancer and be a useful tool in primary prostate cancer detection suspected relapse or metastatic disease using 11C-acetate and in monitoring the therapeutic effect of an antiand- PET and 15 with F-FDG PET additionally. In total, 14 rogen therapy. A superior sensitivity in the detection of patients received hormonal ablation therapy at the time distant metastases in comparison to 18F-FDG PET was of the scan. not seen.

Prostate Cancer and Prostatic Diseases Abstracts S21 Positive margins after radical prostatectomy in the Instituto Nacional de Cancerologia. Mexico

P Martinez-Cervera, M Aragon-Castro and A Jime´nez-Rios Miguel

Introduction group was from 6 to 110 months with median of 58 months. The PSA level varies from 4.1 to 37.5 According with the literature, the incidence of positive ng/ml. The Gleason score was between 4 and 9. From margins in radical prostatectomy varies from 16 to 46%. the group of positive margins, 10 patients (40%) received In Mexico, there are no data about this found in only surgery as single therapy, 14 patients (56%) received the cancer centers. This study presents the management radiotherapy and one (4%) was included in the CAPRI of the patients and their evolution according with the study. The median follow-up was 58.5 months. The level of Prostatic Specific Antigen (PSA) and Gleason PSA levels vary from 1.3 to 34.5 ng/ml. The Gleason score. score was between 6 and 10. Patients with biochemical evidence of progression were treated with maximal androgen blockade. There was no difference in global Materials and Methods survival between the two groups. There in no evidence of progression in the two patients with positive We reviewed a period of 11 years since January 1992 to surgical margins (both with Gleason score 47) treated January 2002, and found a total of 93 radical prostatect- with MAB at 53 month of follow-up. Patients with omy in The Insituto Nacional de Cancerologia Mexico. Gleason score 8–10 treated with radiotherapy, and PSA Then we selected the cases with positive margins and level 420 ng/ml in the group with negative surgical positive surgical borders. The patients were divided in 3 margins showed a less progression-free time in each groups according with the adjuvant therapy they group. At this time, 92% of the patients are alive without received based in the pathological findings (Gleason evidence of tumoral activity, one patient (4%) is death score) and the PSA level at diagnosis. by cancer and one (4%) died for cardiac disease without tumoral activity. Results We found a total of 40 patients that represents the 44.4% Discussion of the total of patients treated with radical prostatectomy, 15 (37.5%) had positive surgical borders, and 25 (62.5%) The findings in this study are very similar than had positive margins. Multiple capsular penetration others reported in the literature, we cannot suggest and seminal vesicles were the most common site of that radiotherapy must be adopted as a universal invasion, and the apex was the first place of positive adjuvant therapy, but it must be considered in patients surgical margin. From the group of positive surgical with surgical positive margins and high Gleason borders, two patients (13.3%) were treated only with scores. On the other hand, patients with risk of surgery, 11 patients (73.3%) received adjuvant radio- progression could be treated with hormonal therapy, therapy and two (13.3%) patients were treated with and the intermittent therapy should be considered as an maximal androgen blockade. The follow-up for this option.

Prostate Cancer and Prostatic Diseases Abstracts S22 Mutation analysis of the androgen receptor gene among Finnish prostate cancer patients

MP Matikainen1,3, ER Hyytinen2, T Ikonen1, TL Tammela3, JSchleutker1 and PA Koivisto1,2 1Laboratory of Cancer Genetics, Tampere University Hospital, Tampere, Finland 2Department of Clinical Genetics, Tampere University Hospital, Tampere, Finland 3Department of Urology, Tampere University Hospital, Tampere, Finland

Background androgen receptor gene using single-strand conforma- tion polymorphism analysis (SSCP) and subsequent Most prostate cancer (CaP) cases are sporadic but strong sequencing. genetic and epidemiological evidence indicate that CaP also has a hereditary component. Recent studies have provided epidemiological support of a possible Results role of androgen receptor (AR) gene as prostate cancer susceptibility locus. SSCP screening of the coding region of the AR gene showed band shifts in 10 of the 74 (13.5%) CaP patients. A silent polymorphism Q211Q in exon 1 was found in Objectives two (2/38, 5.3%) early-onset CaP patients and in seven affected members of HPC families (7/36, 19.4%). One To study the role of androgen receptor (AR) gene R726L substitution in exon 5 was found in one prostate mutations in CaP, the coding region of the AR gene cancer family, but no previously uncharacterized muta- was screened for mutations using genomic DNA of tions were detected. familial and early-onset CaP patients.

Conclusions Methods To our knowledge, our present study is the first to report DNA samples from 38 early-onset (range 47–59 y) the comprehensive AR gene mutation screening prostate cancer patients with negative family history in familial and early-onset CaP patients. Our results for malignancies and 36 patients from Finnish prostate indicate that androgen receptor gene mutations are not cancer families showing no male-to-male transmission of common factors influencing the genetic predisposition prostate cancer were analyzed for mutations in the of CaP.

Prostate Cancer and Prostatic Diseases Abstracts S23 Invasive characterisitics of human prostatic epithelial cells

IG McIntyre1, CA Hart1, MD Brown1, LJ Scott1 and NW Clarke1 1Department of Urology, Christie Hospital, Manchester, UK

Bone metastases are the major cause of morbidity and cells showed that CaP epithelial cells were invasive, mortality for patients with prostate cancer. We preferentially towards bone marrow stroma and pro- have previously reported that prostate epithelial cells static fibroblasts, while BPH epithelial cells were non- (PEC) isolated from both benign prostatic hyperplasia invasive. (BPH) and carcinoma of the prostate (CaP) patients bind Confocal microscopy of PC3-GFP (GFP expressing) preferentially to and at equivalent levels to bone marrow invasion through a bone marrow endothelial cell endothelium. However, only CaP epithelial cells have an layer (BMECs) revealed that 80% of PEC cells bound invasive response towards bone marrow endothelium. within 60 min and all cells bound by 90 min. Binding The aim of this study was to analyse the invasive and invasion occurred at the junctions between BMECs. characteristics of malignant prostate epithelial cells. Invasion through the BMECs was complete 182743 min In an invasion chamber assay, bone marrow endothe- post binding. lium, bone marrow stroma and their extra cellular We have shown that both bone marrow endothelium matrixes induced the migration of the malignant PC-3 and stroma induced malignant prostate epithelial cells to cell line, while the nonmalignant cell line PNT2-C2 invade. Binding and invasion occurred at the cellular demonstrated no invasive ability. Invasion chamber junctions between bone marrow endothelial cells, with assays using primary BPH or CaP prostate epithelial the invasive process taking approximately 180 min.

Prostate Cancer and Prostatic Diseases Abstracts S24 DD3PCA3-based molecular urine analysis for the diagnosis of prostate cancer

P Mulders1, D Hessels1, B Van Balken1, H Karthaus1 and J Schalken1 1Department of Urology, University Hospital Nijmegen, The Netherlands

Background data revealed that specimens with less than 10% of cancer cells could be accurately discriminated from DD3PCA3 is the most prostate cancer-specific gene noncancer tissues. Hence, detection of a small fraction described to date. To assess the clinical utility of DD3PCA3 of prostate cancer cells in a background of normal a time-resolved fluorescence based, quantitative RT-PCR cells seemed feasible. Therefore, this DD3PCA3-based analysis for DD3PCA3 was developed. RT-PCR assay was used for the identification of pro- state cancer in urine sediments obtained after prostatic massage. From 108 men with a serum PSA value 4 Methods 3 ng/ml, 24 men were shown to have prostate cancer upon biopsy. Of these 24 men, 16 were shown to be PCA3 The diagnostic potential of DD3 was determined positive for DD3PCA3, indicating a sensitivity of the assay PCA3 by quantitative measurement of DD3 transcripts of 67%. Furthermore, a negative predictive value of 88% in nonmalignant and malignant prostate specimens. was calculated. Moreover, DD3PCA3 transcripts were determined quanti- tatively in urine sediments obtained after prostatic massage. A cohort of 108 men, admitted for prostate biopsies based on a PSA of 43 ng/ml, was studied. Conclusion Results The quantitative RT-PCR assay for DD3PCA3 described bears great promise as a tool for molecular urine Prostate tumors showed a 66-fold upregulation of analysis. It has great potential in reducing the number DD3PCA3 (median 158.4 Â 105 copies/mg tissue) when of unnecessary biopsies. A multicenter study using compared to benign prostate tissue (median 2.4 Â 105 this DD3PCA3 assay can provide the first basis for copies/mg tissue). This upregulation was found in more the utility of molecular diagnostics in clinical urological than 95% of prostate cancer specimens studied. These practice.

Prostate Cancer and Prostatic Diseases Abstracts S25 Development of prostate cancer gene therapy as a translational research: what we have achieved and where we have to go

Y Nasu1, H Kumon1 and TC Thompson2 1Okayama and University, Okayama, Japan; and 2Baylor College of Medicine, Houston, TX, USA

Objectives system. The antitumoral properties of interleukin (IL-12) have been demonstrated in multiple preclinical In order to elucidate current status and future prospect studies. In our preclinical (mouse) trials, adenovirus- of prostate cancer gene therapy, our both basic and mediated expression of mouse IL-12 was used to treat clinical outcomes are reported and future aspect of a prostate cancer model we developed. We demonstrated prostate cancer gene therapy as an effective clinical a significant reduction in prostate tumor weight alternative for hormone refractory prostate cancer is and suppression of pre-established lung and bone discussed. metastasis as well as a survival advantage in IL-12 virus treated mice (Gene Therapy 1999; 6:338). Clinical protocol is already approved in USA and under review Clinical results and its implication in Japan. In our previous studies, we demonstrated the efficacy and safety of herpes simplex virus-thymidine kinase Basic research outcome and its clinical gene transduction and ganciclovir therapy in an ortho- topic mouse model of prostate cancer (Hum Gene Ther application 1996; 7:515.). These preclinical studies resulted in the In order to further investigate new target molecule and initiation of clinical trials in USA (Baylor College of expand our translational research for prostate cancer Medicine) for those patients who have local recurrence gene therapy, our group has been conducting differential without clinical metastasis after definitive radiation gene profile analysis in immortalized cells. Recently, therapy and in Japan (Okayama University) for patients it is demonstrated that Reduced Expression in Immorta- who have local recurrence after hormonal therapy. Safety lized Cell (REIC) gene is a new candidate of tumor and positive clinical response were shown (Hum Gene suppressor gene, because of its low expression in human Ther 2001; 12:1955.). cancer cell lines including prostate cancer and renal cell carcinoma. Development of new clinical trial for advanced prostate cancer Conclusion These efforts as well as preclinical experimental studies Prostate cancer gene therapy as a clinical alternative for with mice have led us to conclude that a necessary hormone refractory and metastatic disease is still at an component of any successful gene therapy approach early stage in its development. Several technological directed against prostate cancer that may have spread breakthrough are required, but some favorable results to other parts of the body (metastases) must include obtained from some clinical trials warrant full potential a component that activates the host antitumoral immune for wide scale of practice.

Prostate Cancer and Prostatic Diseases Abstracts S26 Comparison of three different methods of anesthesia during transrectal prostate biopsy: a prospective randomized trial

CO¨ bek1,BO¨ zkan1, B Tunc1, G Can1, V Yalcin1 and V Solok1 1University of Istanbul Cerrahpasa School of Medicine, Istanbul, Turkey

Introduction taken from each patient. Pain was assessed upon completion of the procedure with a numeric analogue Two factors are held responsible for pain during prostate scale. Complications were noted. Four groups were biopsy: anal discomfort of the ultrasound probe compared regarding the severity of pain/discomfort. and insertion of needles through the prostate gland. Complications were also compared. Kruskal–Wallis and Periprostatic nerve block and intrarectal lidocaine gel w2 tests were used for statistical analysis. application have both been shown to decrease pain and discomfort during transrectal ultrasound (TRUS)-guided prostate biopsies. A randomized trial suggested that Results needle puncture for nerve block was more painful than the biopsy itself. In addition, periprostatic infiltra- Each group consisted of 75 patients. Median age, tion was shown to be associated with a higher infectious prostate volume and PSA values were similar among complication rate due to extra needle punctures. the groups. A comparison of pain scores showed A method that is free of extra needle punctures would statistically significance among them (Po0.0001). overcome both of these shortcomings. We hypothesized Median pain scores were 4.63 in controls, 2.57 in that the addition of rectal/perianal lidocaine gel applica- periprostatic infiltration group, 2.03 in infiltration þ gel tion may serve two-fold: (1) decrease pain from the group, and 3.11 in tramadol HCl group. Thus, any probe, and (2) decrease pain from needle puncture kind of anesthesia was superior to controls. Pain of nerve block. Tramadol HCl, a codeine derivative was least in periprostatic infiltration þ gel group. with centrally acting analgesia, was used intravenously The difference of pain score between periprostatic in another group of patients as a noninvasive method. nerve blockade alone and tramadol groups did not To our knowledge, both these anesthesia techni- reach statistical significance. Complication rates were ques have never been used before. The two novel similar. methods were compared with each other and with the ‘standard’ periprostatic nerve block as well as with a control group. Conclusions (1) Any form of analgesia/anesthesia used in this study Material and method proved to be superior to controls. (2) The combination of periprostatic nerve block and A total of 300 patients who underwent TRUS-guided lidocaine gel provided significantly better analgesia prostate biopsies were randomized into four groups. than the other two methods. If this can be duplicated Group 1 served as the controls without any anesthesia/ in other trials, it should be accepted as the new gold analgesia. Periprostatic nerve infiltration with 2% standard. lidocaine, 2.5 cm3. to both sides of the prostate, (3) Although the efficacy of tramadol was slightly was performed in Group 2. In Group 3, perianal/ inferior to periprostatic nerve block, the difference intrarectal application of 10 cm3 2% lidocaine gel failed to reach a statistical significance. Thus, was used in addition to the periprostatic infiltration. tramadol could be a viable alternative form of Tramadol HCl was infused intravenously in 30 min at a analgesia/anesthesia for prostate biopsies and needs dose of 1.5 mg/kg/100 cm3 saline. Twelve cores were to be explored in future studies.

Prostate Cancer and Prostatic Diseases Abstracts S27 Detection and clinical implications of lymph node occult metastasis in patients with pN0 prostate cancer

V Pagliarulo1, D Hawes2, SL Groshen2 and RJ Cote2 1Sezione di Urologia, DETO, Universita` degli studi di Bari, Italy; and 2Department of Pathology, University of Southern California, CA, USA

Introduction for a total of 23/2820 lymph nodes resulting positive (0.8%). For all positive cases, the prostatic origin of Over the last decade, our group and others have the epithelial cells was confirmed by PSA staining; the explored the possibility of identifying undiagnosed original hematoxylin and eosin stained sections were disease in the LN removed from prostate cancer patients, also retrieved, and in three cases a careful review by means of an immunohistochemical approach. Thus, would confirm the presence of tumor cells. None of we have studied the incidence of occult LN metastasis the demographic features were significantly associated (OLM), after radical prostatectomy and bilateral pelvic with LN status. However, patients with OLM were lymph node dissection, in a cohort of 141 pT2b-T3b N0 more likely to have stage pT3b, high grade disease patients diagnosed with adenocarcinoma of the prostate. (Gleason score 7–10), and positive margins, as compared With a median follow-up time of 11.5 y, we have to LNÀ patients. compared the clinical outcome in patients with negative By 10 y, the estimated survival, time to clinical nodes, occult metastasis, and, finally, with a cohort of 91 recurrence, and time to any recurrence probabilities positive nodes (N þ ) patients. were all lower for LN þ and for OLM patients compared to LNÀ disease. By 10 y, the estimated probability of dying for LNÀ, OLM, and LN þ patients, was Methods 0.2170.04, 0.5070.12, and 0.3770.06, respectively; the estimated probability of having a clinical recurrence Formalin-fixed paraffin-embedded tissue blocks of was 0.1670.03, 0.3170.12, and 0.3570.05 for patients lymph node specimens were retrieved for the 141 con- with LNÀ, OLM, and LN þ patients; and the secutive patients. A total of 1015 blocks (7.2 per patient), estimated probability of experiencing any recurrence and 2820 lymph nodes (20 per patient) were examined. was 0.4070.05, 0.5470.12, and 0.7270.05, respectively. All blocks containing lymph nodes were serially sec- Relative risks (RR) based on the hazards ratio are tioned at two levels, with 3, 5-mm sections being cut at also examined. Overall and on average, patients with each level. A single section from level 1 was tested with a OLM disease were 2.65 times more likely to die cytokeratin antibody cocktail, using a common immu- (P ¼ 0.003), 1.97 times more likely to have a clinical noperoxidase protocol. The slides were reviewed by recurrence (P ¼ 0.16), and 1.66 times more likely three investigators in a blind fashion. If cytokeratin- to experience any type of recurrence (P ¼ 0.14), compared positive cells were identified, a second adjacent section to patients with LNÀ disease. In a multivariable from level 1 was tested with antibody to PSA. analysis, these relative risks remained essentially unchanged, after stratifying by age, Gleason score, Results TMN staging, and surgical margins: RR ¼ 2.32 and P ¼ 0.006 for survival, RR ¼ 2.00 and P ¼ 0.14 for Occult metastases were found in 18/141 patients (13%), clinical recurrence, and RR ¼ 2.49 and P ¼ 0.20 for any previously believed to be free of lymphatic disease, recurrence.

Prostate Cancer and Prostatic Diseases Abstracts S28 Phase-I Study: cancer gene therapy of hormone refractory prostate cancer patients with a retrovirally transfected IL-2-INF-c secreting allogeneic tumor vaccine

R Paul1, H Kuebler1, H van Randenborgh1, J Breul1, R Hartung1 F Fend2, H Pohla3, D Schendel3, T Brill3 and B Gaens bacher3 1Department of Urology, Technische Universitaet Munich, Germany; 2Institute of Pathology, Technische Universitaet Munich, Germany; 3Institute of Molecular Immunology, GSF, Munich, Germany; and 4Department of Experimental Oncology, Technische Universitaet Munich, Germany

Background progression and ELISPOT and tetramere staining for immunological response. Toxicity was evaluated by Interleukin-2 (IL-2) is used in several gene therapy standard toxicity criteria (NCI CTC 1999). protocols to stimulate IL-2 receptor positive cells like T- cells, natural killer cells and macrophages and enhance their ability to kill tumor cells. Interferon-g (INF-g)induces Results expression of MHC-I molecules, enhanced immunogeni- city and therefore increased immunostimulation. Three patients received 7.5 Mio cells/vaccination. None of these three patients demonstrated a toxicity 4grade 1. One of three patients with dose escalation to 15 Mio Objectives cells/vaccination demonstrated a grade 2 dermal reac- tion at the vaccination site (pain, swelling, inflammation) We propose to study whether immunization with the after the 5th and a transient and self-limiting grade 3 allogeneic HLA class I matched prostate carcinoma cell toxicity (ulceration) after the 8th vaccination. There was line LNCaP expressing recombinant IL-2 and INF-g will no dose limiting or autoimmune toxicity seen. We found be tolerated and will be able to induce an immune a trend towards a prolonged median time to disease response in hormone refractory prostate cancer. progression for patients with the higher dose.

Methods Conclusions Six patients with hormone refractory prostate cancer Our results demonstrate that a gene therapy with were treated with four vaccinations on day 1, 15, 29 and retrovirally transfected IL-2-INF-g-secreting allogeneic 90 of irradiated, allogeneic LnCAP-cells secreting IL-2 tumor vaccine is generally well tolerated. In an ongoing and INF-g by retroviral gene transfer. Vaccination was phase-II-trial, we are evaluating the efficacy of this continued every 3 months until tumor progression. protocol for cancer control in hormone refractory Monitoring was performed by PSA, bone scan for tumor prostate cancer.

Prostate Cancer and Prostatic Diseases