Prostate Cancer

REVIEWING THE LITERATURE News and Views from the Literature Prostate Cancer Peripheral Blood RT-PCR Assays for Detection and Prognosis of Prostate Cancer Reviewed by Shahrokh F. Shariat, MD,* Kevin M. Slawin, MD† *Department of Urology, University of Texas Southwestern Medical School, Dallas, TX; †The Baylor Prostate Center, Scott Department of Urology, Baylor College of Medicine, Houston, TX [Rev Urol. 2003;5(1):54–58] © 2003 MedReviews, LLC he application of molecular biology in medicine has led to new, potentially powerful modalities for the diagnosis and staging of cancer. For example, reverse transcriptase polymerase chain reaction (RT-PCR) has been investigated as a potentially new diagnostic and staging modality for prostate cancer. The RT-PCR assay is a relatively simple and rapid, yet sensitive and specific technique that amplifies a targeted, tissue-specific messenger ribonucleic acid (mRNA) sequence. RT-PCR makes possible the detection of small quantities of cells that are currently undetectable in peripheral blood or within other body fluids or tissues. Since Moreno and associates1 and Vessella and associates2 pioneered the use of RT-PCR for the detection of circulating occult prostate cells, there has been a rapid increase in the number of reports in the literature about the use of RT-PCR for detecting cells expressing prostate-specific antigen (PSA) and other prostatic markers, such as prostate-specific membrane antigen (PSMA) and human glandular kallikrein (hK2). This new assay technology has the potential to allow development and assessment of new therapeutic strategies and to help monitor disease activity more accurately. In cases of localized prostate cancer, it could help identify patients T 54 VOL. 5 NO. 1 2003 REVIEWS IN UROLOGY who harbor occult micrometastases, sparing them the morbidity of a radical prostatectomy or radiation therapy that would be ineffective, and/or help select those patients best suited for clinical trials of neo-adjuvant and/or adjuvant therapy. However, while foci of metastatic prostate cancer detectable by conventional modalities (eg, histology, bone scan, or computed tomography scan) are almost always associated with biologically significant advanced disease, the presence of prostate cells in the circulation does not necessarily indicate that clinically significant metastasis has occurred. Therefore, RT-PCR technology places the burden squarely on investigators to demonstrate the clinical and biological significance of a positive result. Some studies have demonstrated a significant predictive value in RT-PCR for PSA or PSMA for both the pathologic stage of prostate Newer, ultrasensitive assays, like RT-PCR, that can identify small numbers of cells, place the burden squarely on investigators to demonstrate the clinical and biological significance of a positive result. cancer3–6 and progression-free survival.7-9 However, the majority of studies have failed to demonstrate any clinically significant role for RT-PCR for PSA or PSMA.10-14 The limited follow-up time and formidable technical problems regarding sample processing and handling may underlie these conflicting results. Recently, Klein and associates (article 1) and Slawin and associates (article 2) reported on the absence of any association between RT-PCR for PSA and/or PSMA and clinical and pathologic characteristics and biochemical progression in patients who underwent radical prostatectomy for clinically localized disease and who had long-term prospective follow-up (median of 31 and 53 months, respectively). Interestingly, Slawin and his Prostate Cancer team did find that RT-PCR detection of the native hK2 protein was associated with features of biologically aggressive prostate cancer and clinical outcome (article 2). These studies further emphasize the importance of targeting prostate-specific markers that are associated with biologically and clinically aggressive disease. One possible limitation of most studies to date is that they have relied on the simple qualitative detection of PSA-expressing cells or on semiquantitative techniques, even if measures were taken to increase the sensitivity by using nested PCR techniques or radioactive detection methods. Because target amplification by PCR is exponential in nature, these approaches show no linearity of reaction to the amount of prostatic target mRNA applied. Moreover, virtually all quantitative detection methods published thus far have the disadvantage of not being easily adapted for standardized clinical application in many centers. To overcome these limitations, Ylikoski and associates recently developed highly sensitive, quantitative, internally standardized RT-PCR assays for PSA15 and hK2,16 and evaluated their utility for diagnosis and staging of prostate cancer (article 3). In view of the findings of the reviewed articles, and the literature in general, the consensus is that at the present time RT-PCR technology is not ready for use in the clinical management of patients with prostate cancer. However, this molecular test is still highly promising. Technological advances such as immunomagnetic enrichment and digital microscopy, among others, may improve the assays and lead to a quantitative, reproducible assay that can specifically detect those select prostate cancer cells that are associated with the development of clinically evident distant metastases, thereby making them more useful in the clinical setting. Preoperative Combined Nested Reverse Transcriptase Polymerase Chain Reaction for Prostate-Specific Antigen and ProstateSpecific Membrane Antigen Does Not Correlate with Pathologic State of Biochemical Failure in Patients with Localized Prostate Cancer Undergoing Radical Prostatectomy. Thomas J, Gupta M, Grasso Y, et al. J Clin Oncol. 2002;20:3213–3218. In 19975 and 1998,6 Klein and associates developed nested RT-PCR assays for PSA and PSMA and reported on their preliminary results. They tested the hypothesis that the combination of these two markers would increase the sensitivity of their assays by partially overcoming the problem of tumor cell heterogeneity. They found that their RT-PCR assay for PSMA was consistently more sensitive than that for PSA, yielding more than 1.8–3 times the number of positive results. However, the combination of both assays together (PSA and/or PSMA positive vs both negative) was superior to either assay alone in sensitivity and specificity. Like most reported assays, the lower end of detection of their assays was one LNCaP cell diluted in 106 mononuclear cells. The clinical specificity of their combined assay was 100%, yielding no false-positives in normal subjects (n = 15), female subjects (n = 5), and men with confirmed benign Combined nested RT-PCR for PSA/PSM is not an independent predictor of either pathologic stage or biochemical failure in patients with localized prostate cancer undergoing radical prostatectomy. prostatic hyperplasia (n = 15). The clinical sensitivity of their combined assay was 100% in patients with clinically evident distant metastases (n = 11) and yielded no falsenegative results. Here again the RT-PCR-PSA assay performed suboptimally, yielding only 64% positivity when used alone, while RT-PCR-PSMA detected all except one patient. In both of their initial studies, Klein and associates found that a combined, nested preoperative RT-PCR assay for both PSA and PSMA was an independent predictor of extracapsular extension on radical prostatectomy specimens.5,6 In a well-designed and executed follow-up study, the team at the Cleveland Clinic Foundation evaluated the association of preoperative peripheral blood RT-PCR for PSA and PSMA with pathologic stage and, more importantly, with biochemical progression in 141 patients undergoing radical prostatectomy for clinically localized disease. For quality control they duplicated the assay in the first 25% of patients and found less than 5% discordance in assay results. Overall, 52% of the patients had a positive assay result for PSA and/or PSMA, a rate that is higher than noted in their previous reports and higher than reported in most published studies. In concordance with their previous reports, the large majority of patients were positive for PSMA only, with 5% of patients having a positive assay result for both RT-PCR-PSA and RT-PCR-PSMA and 3% for RT-PCR-PSA only. In contrast with their initial studies, in this larger set of more contemporary patients, the authors failed to find an association between RT-PCR status and pathologic stage. In addition, with a median follow-up of 31 months, RT-PCR status was not associated with disease progression after surgery, even in the subset of patients with non-organ-confined disease. These findings did not change when patients with preoperative androgen-deprivation therapy were excluded (n = 27). The authors conclud- VOL. 5 NO. 1 2003 REVIEWS IN UROLOGY 55 Prostate Cancer continued ed that preoperative combined nested RT-PCR for PSA and PSMA has no clinical utility in patients undergoing radical prostatectomy for clinically localized disease. Preoperative Blood RT-PCR Assays for Prostate-Specific Antigen and Human Glandular Kallikrein for Prediction of Prostate Cancer Progression After Radical Prostatectomy. Shariat SF, Gottenger E, Nguyen C, et al. Cancer Res. 2002;62:5974–5979. The questionable specificity of current markers for prostate cancer cells and the potentially variable biologic and clinical potential of the cells detected by ultrasensitive assays have discouraged the use of PSA and PSMA as targets for RT-PCR assays in the clinical setting.17-20 Therefore, we developed a highly sensitive and specific RT-PCR assay for hK2 mRNA21 that was designed to differentially identify two previously described splice variants of the hKLK2 gene.22 Our primers differentiated between amplification of the native hk2 transcript (hK2-L) that encodes for the full-length hK2 protein, and an alternate spliced transcript (hK2-U) that contains an additional 37 nucleotides downstream from the native splice donor site in intron IV.22 This larger, alternatively spliced mRNA is predicted to encode a truncated and, presumably, nonfunctional version of the hK2 protein. Our assay provided maximum sensitivity and specificity for hK2 without any cross-reactivity to the closely related kallikreins, hK1 and PSA. In addition, our assay was highly sensitive, as demonstrated by a reliable detection of five copies of hK2 cDNA and at least one LNCaP cell in 109 cultured lymphoblasts. When evaluated in peripheral blood specimens obtained from 14 men at low risk for harboring prostate cancer, 14% of the control specimens tested positive for hK2-L. On the other hand, the clinical sensitivity was superior to that reported in the literature for RT-PCRhK2 assays with 86% of the 7 patients with untreated metastatic prostate cancer yielding a positive assay result.23,24 When the assay was performed on peripheral blood of patients with clinically localized prostate cancer prior to radical prostatectomy, the native hK2-amplified fragment, but not the splice variant fragment (hK2-U), was an independent predictor of metastasis to regional lymph nodes.21 On the basis of these observations, we hypothesized that RT-PCR for hK2-L assays would detect circulating cells that might indicate the presence of occult metastatic disease in patients undergoing radical prostatectomy and would therefore be associated with prostate cancer that progressed despite effective control of local disease. In addition, to determine the relationship between pre-operative peripheral 56 VOL. 5 NO. 1 2003 REVIEWS IN UROLOGY blood RT-PCR for PSA and the risk for prostate cancer progression, we studied a large (n = 224) cohort of consecutive patients with clinically localized prostate cancer who underwent radical prostatectomy and who had long-term follow-up (median follow-up of 53 months, range 1.3–73). Our assay for PSA was highly sensitive, as demonstrated by a reliable detection of five copies of PSA cDNA and at least one LNCaP cell in 106 cultured lymphoblasts, but less sensitive than our assay for hK2. Our RT-PCR assay was clinically more sensitive for detecting PSA-expressing cells in patients with metastatic disease (seven of eight patients positive, 88%) than the average of peripheral blood assays used in previous studies (53%, range 13%–100%).25 RT-PCR-hK2-L is associated with established markers of biologically and clinically aggressive prostate cancer, potentially improving our ability to predict metastasis to regional lymph nodes and disease progression. Only one of 14 control specimens (7%) obtained from men at low risk for harboring prostate cancer tested positive for PSA, which is in the range of false positives reported in the literature.25 Of the peripheral blood specimens obtained from 224 patients with clinically localized prostate cancer, RT-PCR assays for PSA, hK2-L, and hK2-U were positive in 24%, 25%, and 26%, respectively. RT-PCR-hK2-L positivity was associated with higher pathologic Gleason score but not organ-confined disease. This confirmed our previous finding that preoperative peripheral blood RT-PCR-hK2-L is an independent predictor of metastases to regional lymph nodes. While these associations with pathologic features are important, an association with occult metastases that can lead to disease progression in patients treated effectively for clinically localized disease would be more useful for managing patients with prostate cancer. Although in univariable analysis, preoperative RT-PCR-hK2-L was a predictor of disease progression after surgery, when adjusted for the effects for preoperative PSA level, biopsy Gleason sum, and clinical stage, this association was no longer significant. However, the relatively low progression rate (21%) and the high rate of patients with features of non-aggressive failure (59%) in this data set might have limited the statistical power of our study for detecting a predictive effect of RTPCR-hK2-L on overall prostate cancer progression. An improved ability to predict the likelihood of clinical disease progression to metastases would have a greater clinical impact on managing prostate cancer patients. We found Prostate Cancer Table 1 Number of Men with Positive RT-PCR Assay Results Benign Disease Total (n = 19) Prostate Cancer Organ Total Confined (n = 51) (n = 10) NonOrgan Confined (n = 6) Lymph Node Involvement (n = 8) Hormone Responsive Bone Metastases (n = 6) Hormone Independent Clinically Bone Metastases Staged (n = 11) (n = 10) RT-PCR Positive (No. pts, %) PSA 1 (5) 41 (80) 6 (60) 6 (100) 8 (100) 5 (83) 9 (82) 7 (70) hK2 1 (5) 43 (84) 6 (60) 6 (100) 8 (100) 6 (100) 9 (82) 8 (80) PSA and hK2 1 (5) 38 (75) 5 (50) 6 (100) 8 (100) 5 (83) 7 (64) 7 (70) PSA or hK2 1 (5) 46 (90) 7 ( 70) 6 (100) 8 (100) 6 (100) 11 (100) 8 (80) PSA, prostate-specific antigen; RT-PCR, reverse transcription polymerase chain reaction; hK2, human glandular kallikrein. Data from Ylikoski et al.16 that preoperative RT-PCR-hK2-L was associated with features of aggressive prostate cancer progression, defined by a PSA doubling time of less than 10 months,26,27 the failure to respond to salvage local radiation therapy,28 and/or a positive metastatic work-up. This suggests an association of RT-PCR-hK2-L with occult metastatic disease present at the time of radical prostatectomy. Neither RT-PCR for PSA nor hK2-U was associated with any clinical or pathologic characteristics or clinical outcome of patients with clinically localized disease undergoing radical prostatectomy and long-term follow-up. RT-PCR assays for PSA or hK2-U are not useful staging tools for guiding therapy or predicting outcome in patients with clinically localized prostate cancer, and, therefore, they have limited clinical utility. Simultaneous Quantification of Prostate-Specific Antigen and Human Glandular Kallikrein 2 mRNA in Blood Samples From Patients with Prostate Cancer and Benign Disease. Ylikoski A, Pettersson K, Nurmi J et al. Clin Chem. 2002;48:1265–1271. The above reviewed articles support the need for a more objective assay (ie, real-time standardized, quantitative PCR format) that analyzes product quantity during the logarithmic phase of the reaction, before the plateau, and ideally includes internal controls that co-amplify with the same efficiency as the target molecule. Ylikoski and associates have recently developed the first multiplexed, rapid, and reproducible method that allows simultaneous exact linear quantitation of PSA and hK2 gene expression from the same peripheral blood samples.16 The method uses two target-molecule-like, exogenous, internal standard mRNAs that differ only by two base pairs deleted from their target mRNAs. The similarity in sequence of the internal standards and the targets themselves (PSA and hK2) allow their coamplification with the same primers but still permit distinction in the target amplification product. In addition, the internal standards match the targets in amplification efficiency and product size. A known quantity of synthetic internal standard mRNAs are added into each cell pellet at the beginning of the mRNA extraction and are co-amplified in the RT-PCR reaction with the target mRNAs in the sample, providing a standardized correction for any variations allowing reproducible quantification of the targets. PSA and hK2 and their respective internal standards amplification products are detected with fluorescent europium chelate labels and with detection probes in a microtitration, wellbased hybridization assay. The quantification of PSA and hK2 mRNAs is based on the use of an external calibration curve that compares the target with the internal standard fluorescence ratio in the sample to the ratio in the calibration curve. The assay detected 50 copies of hK2 and PSA mRNA and at least one PSA-expressing and 10 hK2-expressing LNCaP cells in 2.5 x 106 hK2- and PSA-negative cells.16 The functional detection limit of the RT-PCR assay was 100 copies of hK2 or PSA mRNA with an interassay coefficient of variation of less than 23%. Preliminary evaluation in peripheral blood showed that the number of hK2 mRNA copies was significantly higher than the number of PSA VOL. 5 NO. 1 2003 REVIEWS IN UROLOGY 57 Prostate Cancer continued mRNA copies in prostate cancer patients with biochemical progressive disease (n = 4), and with locally advanced and metastatic disease to regional lymph nodes and bones (n = 13). In a cross-sectional study, Ylikoski and associates further evaluated the staging ability of their assay in heterogeneous groups of prostate cancer patients and men with benign disease. The clinical specificity of the assay for detection of prostate cancer was 95%, with 1 of 19 men having benign disease (5%) that yielded a false-positive assay result (Table 1). However, two of the men with benign disease had a prostate biopsy for suspected prostate cancer; one had high-grade intraepithelial neoplasia, and one had prostatitis. The man with the positive PSA and hK2 mRNA assay result had a very low free PSA, for which he had twice undergone biopsy. PSA and hK2 mRNA significantly discriminated between prostate cancer and benign disease (Table 1). Sixty percent of patients with organ-confined cancer (n = 10) had a positive assay for PSA and hK2 mRNA. This is the highest rate reported in the literature for patients with organ-confined prostate cancer.25 Within patients with organ-confined disease, PSA and hK2 mRNA positivity was significantly associated with a higher Gleason score. This association was, however, not significant when tested in all prostate cancer patients. PSA and hK2 mRNA was detected in all non–organ-confined (n = 6) and pelvic lymph node–positive (n = 8) patients. In addition, PSA and hK2 mRNA were detected in 83% and 100%, respectively, of patients with distant, hormone-responsive bone metastases (n = 6). In patients with hormone-independent cancer (n = 11), PSA and hK2 mRNAs were each detected in 82% of patients, whereas only 64% were positive for both. There was no association between PSA or hK2 mRNA copy numbers and different disease stages. However, the small sample size of the study limited the statistical power for detecting such association. In addition, the study design and the absence of follow-up do not allow conclusions about the clinical utility of this promising new assay. We are awaiting prospective evaluation of the clinical utility of this assay in a consecutive patient cohort with longterm follow-up. References 1. 2. 3. 4. 5. Moreno JG, Croce CM, Fischer R, et al. Detection of hematogenous micrometastasis in patients with prostate cancer. Cancer Res.1992;52:6110–6112. Vessella R, Blouke K, Stray J. The use of polymerase chain reaction to detect metastatic prostate cancer cells in lymph nodes and bone marrow [abstract]. Proc Am Assoc Cancer Res. 1992;33:396. Ghossein RA, Scher HI, Gerald WL, et al. Detection of circulating tumor cells in patients with localized and metastatic prostatic carcinoma: clinical implications. J Clin Oncol. 1995;13:1195–1200. Katz AE, de Vries GM, Benson MC, et al. The role of the reverse-transcriptase polymerase chain reaction assay for prostate-specific antigen in the selection of patients for radical prostatectomy. Urol Clin North Am. 1996;23:541–549. Zhang Y, Zippe CD, Van Lente F, et al. Combined nested reverse transcription- 58 VOL. 5 NO. 1 2003 REVIEWS IN UROLOGY 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. PCR assay for prostate-specific antigen and prostate-specific membrane antigen in detecting circulating prostatic cells. Clin Cancer Res. 1997;3:1215–1220. Grasso YZ, Gupta MK, Levin HS, et al. Combined nested RT-PCR assay for prostate-specific antigen and prostate-specific membrane antigen in prostate cancer patients: correlation with pathological stage. Cancer Res. 1998;58:1456–1459. de la Taille A, Olsson CA, Buttyan R, et al. Blood-based reverse transcriptase polymerase chain reaction assays for prostatic specific antigen: long term follow-up confirms the potential utility of this assay in identifying patients more likely to have biochemical recurrence (rising PSA) following radical prostatectomy. Int J Cancer. 1999;84:360–364. Okegawa T, Nutahara K, Higashihara E. Preoperative nested reverse transcription-polymerase chain reaction for prostate specific membrane antigen predicts biochemical recurrence after radical prostatectomy. BJU Int. 1999;84:112–117. Mejean A, Vona G, Nalpas B, et al. Detection of circulating prostate derived cells in patients with prostate adenocarcinoma is an independent risk factor for tumor recurrence. J Urol. 2000;163:2022–2029. Oefelein MG, Ignatoff JM, Clemens JQ, et al. Clinical and molecular followup after radical retropubic prostatectomy. J Urol. 1999;162:307–310; discussion 310–311. Ellis WJ, Vessella RL, Corey E, et al. The value of a reverse transcriptase polymerase chain reaction assay in preoperative staging and followup of patients with prostate cancer [see comments]. J Urol. 1998;159:1134–1138. Henke W, Jung M, Jung K, et al. Increased analytical sensitivity of RT-PCR of PSA mRNA decreases diagnostic specificity of detection of prostatic cells in blood [see comments]. Int J Cancer. 1997;70:52–56. Ignatoff JM, Oefelein MG, Watkin W, et al. Prostate specific antigen reverse transcriptase-polymerase chain reaction assay in preoperative staging of prostate cancer. J Urol. 1997;158:1870–1874; discussion 1874–1875. Gao CL, Maheshwari S, Dean RC, et al. Blinded evaluation of reverse transcriptase-polymerase chain reaction prostate-specific antigen peripheral blood assay for molecular staging of prostate cancer. Urology. 1999;53:714–721. Ylikoski A, Sjoroos M, Lundwall A, et al. Quantitative reverse transcription-PCR assay with an internal standard for the detection of prostate-specific antigen mRNA. Clin Chem. 1999;45:1397–1407. Ylikoski A, Karp M, Pettersson K, et al. Simultaneous quantification of human glandular kallikrein 2 and prostate-specific antigen mRNAs in peripheral blood from prostate cancer patients. J Mol Diagn. 2001;3:111–122. Dumas F, Gala JL, Berteau P, et al. Molecular expression of PSMA mRNA and protein in primary renal tumors. Int J Cancer. 1999;80:799–803. Levesque M, Hu H, D'Costa M, et al. Prostate-specific antigen expression by various tumors. J Clin Lab Anal. 1995; 9:123–128. Smith MR, Biggar S and Hussain M. Prostate-specific antigen messenger RNA is expressed in non-prostate cells: implications for detection of micrometastases. Cancer Res. 1995;55:2640–2644. Henke W, Jung M, Jung K, et al. Detection of PSA mRNA in blood by RT-PCR does not exclusively indicate prostatic tumor cells [letter]. Clin Chem. 1996;42:1499–1500. Slawin KM, Shariat SF, Nguyen C, et al. Detection of metastatic prostate cancer using a splice variant-specific reverse transcriptase-polymerase chain reaction assay for human glandular kallikrein. Cancer Res. 2000;60:7142–7148. Riegman PH, Vlietstra RJ, van der Korput HA, et al. Identification and androgen-regulated expression of two major human glandular kallikrein-1 (hGK-1) mRNA species. Mol Cell Endocrinol. 1991;76:181–190. Kawakami M, Okaneya T, Furihata K, et al. Detection of prostate cancer cells circulating in peripheral blood by reverse transcription-PCR for hKLK2. Cancer Res. 1997;57:4167–4170. Corey E, Arfman EW, Oswin MM, et al. Detection of circulating prostate cells by reverse transcriptase-polymerase chain reaction of human glandular kallikrein (hK2) and prostate-specific antigen (PSA) messages. PG-184-8. Urology. 1997;50:184–188. Su SL, Boynton AL, Holmes EH, et al. Detection of extraprostatic prostate cells utilizing reverse transcription-polymerase chain reaction Semin Surg Oncol. 2000;18:17–28. Pound CR, Partin AW, Eisenberger MA, et al. Natural history of progression after PSA elevation following radical prostatectomy [see comments]. JAMA. 1999;281:1591–1597. Roberts SG, Blute ML, Bergstralh EJ, et al. PSA doubling time as a predictor of clinical progression after biochemical failure following radical prostatectomy for prostate cancer. Mayo Clin Proc. 2001;76:576–581. Leventis AK, Shariat SF, Kattan MW, et al. Prediction of response to salvage radiation therapy in patients with prostate cancer recurrence after radical prostatectomy. J Clin Oncol. 2001;19:1030–1039. Incontinence Incontinence New Approaches to the Treatment of Overactive Bladder: The Purine Pathways and an Update on the Tension-Free Vaginal Tape Sling Reviewed by John Kim, MD, Michael B. Chancellor, MD Department of Urology, University of Pittsburgh School of Medicine, Pittsburgh, PA [Rev Urol. 2003;5(1):59–60] © 2003 MedReviews, LLC t is no secret that one of the fastest growing areas in urology is pharmacotherapy of the overactive bladder. Worldwide sales of drugs to treat the overactive bladder will soon exceed one billion dollars per year. In the United States there are two good drugs approved for treatment of overactive bladder: controlled-release oxybutynin and tolterodine. We believe that urology is only at the inception point of exciting new developments in the quest for continence. There are many new and exciting drugs in the pipeline. In this literature review we will introduce a concept that probably most urologists have not considered before: manipulation of the purine pathway. In addition, we will provide an update on the tension-free vaginal tape (TVT) sling with some very interesting recent European data. I P2X Receptors and Their Role in Female Idiopathic Detrusor Instability O’Reilly BA, Kosaka AH, Knight GF, et al. J Urol. 2002;167:157–164. Contractions of normal human bladders are primarily induced by acetylcholine (ACh) released from cholinergic nerve terminals in the bladder. However, nonadrenergic, noncholinergic bladder contractions induced by adenosine triphosphate (ATP) increase in the human bladder under pathological conditions, such as denervation, bladder outlet obstruction, or idiopathic urge incontinence. ATP acts on two families of purinergic receptors: an ion channel family (P2X) and a G protein–coupled receptor family (P2Y). P2X receptors appear to be functionally relevant in the bladder, and seven P2X subtypes have been identified. Two subtypes are of particular interest: P2X2 and P2X3. P2X3 receptors have been identified in small-diameter afferent neurons in dorsal root ganglia and in the wall of the bladder and ureter in a suburothelial plexus of afferent nerves. Desensitization of purinergic receptors by intravesical administration of ATP or administration of a receptor antagonist, suramin, depressed bladder afferent activity. In P2X3 knockout mice, afferent activity induced by bladder distension was significantly reduced. This indicates that purinergic receptors are involved in mechanosensory signaling in the bladder and that the loss of P2X3 receptors leads to bladder hypoactivity. In their article, O’Reilly and colleagues reported on 20 women with urodynamics-verified idiopathic detrusor instability who were compared with 20 age-matched control women with urodynamically proven stable bladder. All of the women underwent endoscopic bladder biopsies of the posterior wall. The muscle tissue was analyzed in organ baths for functional studies and the purinergic contribution to its contractility. In addition, quantitative analyses were performed using reverse transcriptase polymerase chain reaction and immunohistochemical localization of P2X receptors. In patients with idiopathic detrusor instability, detrusor P2X2 receptors were significantly increased, whereas other P2X receptor subtypes were significantly decreased. There was no detectable purinergic component of nerve-mediated detrusor muscle conrtractility in normal women without instability. However, there was a significant (approximately 80%) purinergic contractility component in the bladders of women with detrusor instability. In conclusion, the purinergic pathway may be a novel target for the pharmacologic treatment of overactive bladder. Agents acting on the P2X receptor could modulate efferent/afferent activities in treating overactive bladder if receptor subtype-specific agents become available. Drugs that act on P2X2 receptors associated with bladder muscle hold promise for the treatment of detrusor instability. Drugs that act selectively on P2X3 receptors associated with bladder afferent nerves hold promise for the treatment of sensory urgency and decreased compliance. Does the Tension-Free Vaginal Tape Procedure Affect the Voiding Phase? Pressure-Flow Studies Before and 1 Year After Surgery Sander P, Møller LMA, Rudnicki PM, Lose G. BJU Int. 2002;89:694–698. The object of this study from the University of Copenhagen, Denmark was to look at the potential for TVT procedure for stress incontinence to lead to the development of urethral VOL. 5 NO. 1 2003 REVIEWS IN UROLOGY 59 Kidney Stones obstruction. One year after the TVT procedure, 45 treated women were asked if their voiding had changed after surgery. Uroflowmetry, residual urine measurements, and pressure-flow data were also evaluated. The TVT procedure is a minimally invasive surgical method for treating female urinary stress incontinence that has gained widespread popularity. The procedure requires positioning of a vaginal tape precisely underneath the mid-urethra with no tension, to restore urethral support. A sling properly placed mid-urethrally may be obstructive because the tension is too high or the direction of traction inappropriate. The cure rate is comparable with fascial sling techniques used previously. Voiding dysfunction is a well-recognized complication when suburethral sling procedures are used. One of the initial promises of TVT is that it does not cause obstruction. However, the effect of TVT on the voiding phase has been insufficiently assessed until this study. One year after surgery, 39 women (87%) were subjectively cured and 6 (13%) improved. The objective cure rate was 88%. Subjectively, 78% of the patients reported that the voiding phase had become more difficult, and the spontaneous flow curve changed to a more obstructive pattern in 40%, with the mean urinary peak flow rate and the mean average flow rate decreasing significantly. The residual urine volume increased significantly, although no patient had volumes of > 25% of their bladder capacity. During the pressure-flow study, the maximum flow rate decreased and the urethral resistance factor increased significantly. However, only 1 patient could be classified as obstructed. Two patients required intermittent selfcatheterization once daily 1 year after surgery. This study indicates that TVT affects the voiding phase both subjectively and objectively. Information available on voiding phase changes after TVT is scanty. In this prospective study, 3 of 45 (7%) patients developed complete urinary retention after the TVT surgery. About three quarters of the women reported changes in their voiding pattern after surgery, but the significance of these subjective changes is unclear. Residual urine was significantly higher than before surgery, with 2 women having residual urine volume of >100 mL. The present results show that a patient can be severely obstructed without having a significant post-void residual volume, and thus measuring the residual urine in each patient does not provide a reliable means to exclude obstruction. Any operation for stress incontinence may introduce 60 VOL. 5 NO. 1 2003 REVIEWS IN UROLOGY voiding difficulties and changes in voiding function. However, the clinical significance of introducing voiding symptoms, urge incontinence, recurrent urinary tract infections, and objective findings such as elevated residual urine, impaired flow rate/pattern, and changes in pressureflow variables, remains to be elucidated. Currently there is no consensus on how to define voiding difficulty or impaired voiding in terms of obstruction and hypoactive detrusor in women. Consequently, there is no clear threshold level for any voiding phase variable by which intervention (in terms of transection of a sling) is definitively indicated to relieve clinical obstructive symptoms, reduce significant residual urine volume, and eliminate the longterm risk of detrusor insufficiency. Although the TVT procedure has been introduced as “a tension-free vaginal tape," it is clear that the sling has tension applied by the surgeon. Thus, a sling properly placed mid-urethrally may be obstructive because the tension is too high or the direction of traction inappropriate, or because of secondary fibrosis along the sling. In this context, fine-tuning of the tension of the TVT sling, based on coughing with a bladder volume of 300 mL while supine, is arbitrary. Why was 300 mL chosen and not 200 or 326 mL? How scientifically sound is applying the “right amount of tension" to the “tension-free sling" to stop stress incontinence during surgery? Kidney Stones Normal-Calcium, Low-Sodium, and Low-Animal-Protein Diet for Stone Prevention Reviewed by Dean G. Assimos, MD Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC [Rev Urol. 2003;5(1):60–61] © 2003 MedReviews, LLC alcium oxalate nephrolithiasis is influenced by a number of genetic and environmental factors. In the study reviewed below, Borghi and colleagues focused on an important environmental factor, diet, in an effort to reduce stone activity in hypercalciuric, recurrent, calcium oxalate stone formers. C Prostate Cancer Comparison of Two Diets for the Prevention of Recurrent Stones in Idiopathic Hypercalciuria Borghi L, Schianchi T, Meschi T, et al. N Engl J Med. 2002;346:77–84. In this 5-year study, subjects were randomized to a lowcalcium diet, or a normal-calcium, low-sodium, and lowanimal-protein diet. Sixty patients were enrolled in each arm of the study, and a similar proportion (10%) withdrew during the course of the study for various reasons. At the conclusion of the study, the relative risk of stone recurrence in the normal-calcium, low-sodium, and low-animal-protein Oxalate excretion increased in the low-calcium diet subjects but decreased in those on the normal-calcium, low-sodium, and low-animal-protein diet. diet cohort as compared with the low-calcium diet group was 0.49 (P = .04). Calcium excretion significantly decreased in both groups, whereas oxalate excretion increased in the low-calcium diet subjects but decreased in those on the normal-calcium, low-sodium, and low-animalprotein diet. Dietary compliance was excellent in both groups. The relative supersaturation of calcium oxalate decreased significantly from baseline in both groups; however, this was more prominent in the normal-calcium, low-sodium, and low-animal-protein diet group. Subjects with a history of more intense stone activity prior to randomization had diminished risk reduction as compared with those with fewer lead-in stone events. Commentary There were a few weaknesses in this landmark study. The normal-calcium, low-sodium, and low-animal-protein dietary protocol was more tightly regulated. The authors did not provide data on citrate, phosphorus, and uric acid excretion. They also did not analyze differences in the urinary variables between those who had a recurrence and those who did not. The following are take-home messages from this study: 1) an initial trial of a normal-calcium, low-sodium, and low-animal-protein diet should be considered in recurrent, hypercalciuric calcium oxalate stone formers with moderate stone activity; 2) dietary calcium restriction should be avoided in the majority of stone formers, which has also been demonstrated in the studies of Curhan and associates1,2; and 3) patients with more pronounced stone activity may require other measures to attenuate recurrent stone events, such as medical therapy. References 1. 2. Curhan GC, Willett WC, Rimm EB, Stampfer MJ. A prospective study of dietary calcium and other nutrients and the risk of symptomatic kidney stones. N Engl J Med. 1993;328:833–838. Curhan GC, Willett WC, Speizer FE, et al. Comparison of dietary calcium with supplemental calcium and other nutrients as factors affecting the risk for kidney stones in women. Ann Intern Med. 1997;126:497–504. Prostate Cancer Chemoprevention and Prostate Cancer Reviewed by Masood A. Khan, MD and Alan W. Partin, MD, PhD Department of Urology, Johns Hopkins University School of Medicine, Baltimore, MD [Rev Urol. 2003;5(1):61–63] © 2003 MedReviews, LLC rostate cancer is unique in that its behavior may vary greatly between different patients.1 Although prostate cancer may be present histologically in nearly 30% of all men above the age of 50 years, the lifetime risk of developing clinically significant prostate cancer is less than 12%.2,3 Furthermore, the lifetime risk of dying from prostate cancer is less than 4%.2,3 Thus, the development of an effective chemopreventative measure for prostate cancer would greatly affect the natural history of this important disease. Prevention is always better than cure. To this effect, recently there has been a great deal of interest generated in prostate cancer chemoprevention, which is defined as the administration of natural or synthetic agents (pharmaceuticals, biologics, and micronutrients) to prevent initiation, inhibit promotion, or delay progression from normalappearing prostate epithelium to dysplasia to high-grade prostatic intraepithelial neoplasia (HGPIN) to locally invasive adenocarcinoma and metastatic systemic disease.4 The selection of agents to be tested in patients for primary or secondary prostate cancer prevention is based on evidence gathered from epidemiologic, experimental, and clinical studies. Numerous agents are currently under clinical evaluation and may in the future provide a source for preventing the development of clinically significant prostate cancer. Among these agents, selenium, vitamin E, and lycopene hold special interests. A study consisting of more than 29,000 Finnish male smokers revealed that vitamin E (50 IU of dl-alpha tocopherol) was found to be associated with a P VOL. 5 NO. 1 2003 REVIEWS IN UROLOGY 61 Prostate Cancer continued 32% reduction in prostate cancer.5 Another study consisting of more than 1300 men and women at a high risk of developing recurrent nonmelanoma skin cancer revealed that selenium (200 g of selenized yeast) was associated with a marked reduction in the incidence of prostate cancer (67% reduction).6 These studies, despite their limitations and bias, have lead to the decision by the National Institutes of Health to sponsor the Selenium and Vitamin E Chemoprevention Trial (SELECT), which will consist of four arms: selenium versus vitamin E versus selenium and vitamin E versus placebo.7 It is hoped that approximately 32,000 men, with no history of prostate cancer or HGPIN and 55 years of age or older (50 years of age or older if African American), will be recruited into the study from 300 different sites. The study will be conducted for 7–12 years. Lycopene has also generated a great deal of interest, as there is conflicting evidence regarding its association with a reduction in prostate cancer risk.8 The exact mechanism of action of both vitamin E and selenium in tumor prevention is not well established. Two recent studies, reviewed below, shed some light on this matter. The final article reviewed here provides further evidence for the role of lycopene in prostate cancer chemoprevention. Vitamin E Succinate Inhibits the Function of Androgen Receptors and the Expression of Prostate-Specific Antigen in Prostate Cancer Zhang Y, Ni J, Messing EM, et al. Proc Natl Acad Sci U S A. 2002;99:7408–7413. Epidemiological evidence supports the beneficial effect of daily vitamin E supplement in reducing the risk of prostate cancer; however, the underlying mechanism involved is not well established. Using the human prostate cancer cell This study provides credible preliminary evidence for the underlying mechanism of action of vitamin E-mediated inhibition of prostate cancer cells. line LNCaP (derived from lymph node prostate cancer metastasis), the authors have demonstrated that -tocopheryl succinate (VES; 10 M) effectively inhibited the growth of these cells. On the other hand, hydroxyflutamide (HF; 5 M), an antiandrogen, only had a slight inhibitory effect. However, the combination of both VES and HF more significantly inhibited the cells than either agent alone. Using Western and Northern blotting analyses, the 62 VOL. 5 NO. 1 2003 REVIEWS IN UROLOGY authors have also demonstrated that 10 mM of VES effectively repressed prostate-specific antigen (PSA) expression at both the mRNA and protein levels in LNCaP cell cultures. In addition, using Northern blotting they have also shown that VES also suppressed androgen receptor expression by means of transcriptional and post-translational modulation. This VES-mediated inhibition of androgen receptor expression appears to be selective, as VES did not repress the expression of other nuclear receptors. Hence, the authors have concluded that VES may suppress androgen/androgen receptor-mediated cell growth and PSA expression by inhibiting androgen receptor expression at both the transcription and translation levels. This study provides credible preliminary evidence for the underlying mechanism of action of vitamin E-mediated inhibition of prostate cancer cells. This may, in turn, help us to establish new therapeutic concepts for the prevention and treatment of prostate cancer. Selenium Modulation of Cell Proliferation and Cell Cycle Biomarkers in Human Prostate Carcinoma Cell Lines Venkateswaran V, Klotz LH, Fleshner NE. Cancer Res. 2002;62:2540–2545. Venkateswaran and colleagues assessed whether selenium inhibits cell growth and associated cell cycle regulatory proteins. Human prostate cancer cell lines (LNCaP, PC3, PC3-AR2, and PC3-M) were incubated in the presence and absence of selenium (selene-DL-methionine, 150 mM) for 24, 48, and 72 hours. Cells were then fixed and stained with propidium iodide for flow cytometry analysis. Selenium produced a time- and dose-dependent arrest of LNCaP cells in G1 and an 80% reduction in the S-phase. Selenium, on the other hand, had no effect on PC3 cells. However, PC3 cells transfected with the androgen receptor (PC3-AR2) exhibited a G2/M arrest and a marked reduction (57%) in the S-phase during cell cycle progression. Defects in the regulation of cell cycle progression are regarded as common features of transformed cells. Eukaryotic cell cycle progression is regulated by a series of cyclin-dependent kinases, their activity positively regulated by cyclins and negatively regulated by cyclin-dependent kinase inhibitors (CKIs). The significance of growth factors and the signalling pathways mediated by them to regulate the progression of cell cycle in eukaryotes has been identified as an important component of their function. Hence, these authors, using Western blot techniques, also assessed whether inhibition of cell growth was associated with alterations in cell cycle regulatory molecules. Their results Prostate Cancer demonstrated that selenium-treated cells exhibited a timedependent significant up-regulation of CKIs Cip1/p21 and Kip1/p27. In conclusion, this study has shown that selenium possesses strong prostate cancer antiproliferative properties, which appears to be dependent on the presence of a func- The conclusion drawn from this large epidemiological study is that frequent consumption of tomato products is associated with a lower risk of prostate cancer. tioning androgen receptor. The authors have also provided some evidence for the possible underlying mechanisms that may play a role in selenium-associated prostate cancer cell anti-proliferation. A Prospective Study of Tomato Products, Lycopene, and Prostate Cancer Risk period from 1992 through 1998 confirmed their previous findings that frequent tomato or lycopene intake was associated with a reduced risk of prostate cancer.9 Similarly, for the entire period of 1986 through 1998, from taking the cumulative average of the three dietary questionnaires, the authors further determined that lycopene intake was associated with reduced risk of prostate cancer (relative risk [RR] for high vs low quintiles = 0.84; 95% confidence interval [CI] = 0.73-0.96; Ptrend < .001), especially for extraprostatic cancer (RR = 0.65; 95% CI = 0.42-0.99). These associations persisted in analyses controlling for fruit and vegetable consumption, as well as for the use of olive oil. The conclusion drawn from this large epidemiological study is that frequent consumption of tomato products is associated with a lower risk of prostate cancer; however, as the magnitude of this association is only moderate, it is possible to miss this association in a small study. References 1. Giovannucci E, Rimm EB, Liu Y, et al. J Natl Cancer Inst. 2002;94:391–398. 2. 3. In view of the conflicting evidence of the association between tomato products or lycopene, a carotenoid from tomatoes, and prostate cancer risk, these authors evaluated additional data from the Health Professionals Follow-Up Study (HPFS) to further determine whether there is indeed an association between lycopene intake and a reduction in prostate cancer risk. They ascertained prostate cancer cases from 1986 to January 31, 1998, among 47,365 HPFS participants who completed dietary questionnaires in 1986, 1990, and 1994. They found that during the study period 2481 men developed prostate cancer. Results for the 4. 5. 6. 7. 8. 9. Jemal A, Thomas A, Murray T, Thun M. Cancer statistics, 2002. CA Cancer J Clin. 2002;52:23-47. Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics, 1999. CA Cancer J Clin. 1999;49:8-11. Kelloff GJ, Higley HR, Brawer MK, et al. Chemoprevention strategies in the prostate: An overview. Rev Urol. 2002;4:69-77. Lieberman R, Bermejo C, Akaza H, et al. Progress in prostate cancer chemoprevention: modulators of promotion and progression. Urology. 2001;58:835-842. Heinonen OP, Albanes D, Virlano J, et al. Prostate cancer and supplementation with alpha-tocopherol and beta-carotene: incidence and mortality in a controlled trial. J Natl Cancer Inst. 1998;90:440-446. Clark LC, Combs GF, Turnbull BW, et al. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin: a randomised controlled trial. JAMA. 1996;276:1957-1963. Moyad MA. Selenium and vitamin E supplements for prostate cancer: evidence or embellishment? Urology. 2002;59:(4A):9-19. Giovannucci E. Tomatoes, tomato-based products, lycopene, and cancer: review of the epidemiologic literature. J Natl Cancer Inst. 1999;91:317-331. Giovannucci E, Ascherio A, Rimm EB, et al. Intake of carotenoids and retinal in relation to the risk of prostate cancer. J Natl Cancer Inst. 1995;87:1767–1776. VOL. 5 NO. 1 2003 REVIEWS IN UROLOGY 63