Carsten Goessl

Dna-based detection of prostate cancer in urine after prostatic massage

OBJECTIVES Promoter hypermethylation of the glutathione-S-transferase P1 (GSTP1) gene is a specific feature of prostate cancer. This epigenetic DNA alteration served as the target for molecular detection of prostate cancer cells in urine sediments after prostatic massage. METHODS Bisulfite treatment followed by methylation-specific polymerase chain reaction was used to detect GSTP1 promoter hypermethylation in DNA isolated from urine sediments obtained after prostatic massage of men with and without prostate cancer. RESULTS GSTP1 promoter hypermethylation was demonstrated in the sediments of 1 (2%) of 45 patients diagnosed with benign prostatic hyperplasia, 2 (29%) of 7 patients with prostatic intraepithelial neoplasia, 15 (68%) of 22 patients with early, intracapsular cancer, and 14 (78%) of 18 patients with locally advanced or systemic prostate cancer, resulting in a specificity of 98% and an overall sensitivity of 73% for the detection of prostate cancer. CONCLUSIONS Urinalysis for GSTP1 promoter hypermethylation constitutes a sensitive and highly specific DNA-based marker for molecular detection of prostate cancer, including early stages.

DNA‐Based Detection of Prostate Cancer in Blood, Urine, and Ejaculates

Abstract: Background—Methylation‐specific polymerase chain reaction (MSP) targeting promoter hypermethylation of the glutathione S transferase P1 gene (GSTP1), as the most frequent DNA alteration in prostatic carcinoma, was used for the molecular detection of cell‐bound and cell‐free prostate tumor DNA in various human bodily fluids. Materials and Methods—We investigated GSTP1 promoter hypermethylation in DNA isolated from plasma, serum, nucleated blood cells, ejaculates, urines after prostate massage, and prostate tissue from 33 patients with prostate cancer and 26 control patients with benign prostatic hyperplasia (BPH). Using a viral DNA extraction kit specifically recommended for DNA isolation from urine samples, GSTP1 promoter hypermethylation in urine sediments after prostatic massage was investigated in a cohort of 29 patients with prostate cancer and 40 controls with BPH. Fluorescently labeled MSP products were analyzed on an automated gene sequencer. Results—GSTP1 promoter hypermethylation was found in 90% of tumors (18 of 20), 72% of plasma or serum samples (23 of 32), 50% of ejaculates (4 of 8), and between 36% (4 of 11; normal DNA isolation kit) and 76% (22 of 29; viral kit) of exprimated urines from patients with prostate cancer. Also, MSP identified circulating tumor cells in 30% (10 out of 33) of prostate cancer patients. Except for one urine sample, GSTP1 promoter hypermethylation was not found in tissue and body fluids from patients with BPH. Conclusion—GSTP1 promoter methylation analysis provides a highly specific tool for DNA‐based diagnosis of prostate cancer in body fluids.

DNA Alterations in Body Fluids as Molecular Tumor Markers for Urological Malignancies

OBJECTIVES DNA-based tumor markers are characterized by unique specificity rendering them an attractive target for molecular diagnosis of cancer in body fluids like blood serum/plasma and urine. Both cell-free tumor DNA circulating in plasma/serum and cellular tumor DNA are detectable by minimally invasive measures. METHODS Three main detection methods, microsatellite analysis, mutation analysis in genomic or mitochondrial DNA and gene promoter hypermethylation analysis are applied. Detection of gene promoter hypermethylation by methylation-specific PCR enables the best methodical sensitivity requiring a ratio of tumor DNA within normal DNA of less than 1:1000. RESULTS/CONCLUSIONS Tumor DNA derived from renal cell carcinoma, bladder cancer or prostate cancer is detectable in considerably more than 50% of plasma/serum samples and more than 70% of urine samples from these patients. Because the targeted DNA alterations are absent or very rare in controls, the specificity of DNA-based tumor detection methods reaches almost 100%. Although the methodology currently is experimental, automatization will make it easier and less expensive. This review is focused on the potential clinical value of DNA-based analysis of body fluids for the initial diagnosis and the follow-up of urologic cancer patients.

Efficacy and tolerability of tolterodine in children with detrusor hyperreflexia

OBJECTIVES To investigate the urodynamic effects and tolerability of the new antimuscarinic drug tolterodine in children with detrusor hyperreflexia. METHODS Twenty-two children (12 boys and 10 girls; age range 3 months to 15 years, mean age 5.7 years) with detrusor hyperreflexia resulting in maximum detrusor pressures exceeding 40 cm H(2)O during filling cystotonometry were enrolled to receive tolterodine tartrate (a total of 0.1 mg/kg orally daily, divided into two doses) either as a first-line therapy (n = 12, group 1) or replacing oxybutynin chloride therapy (n = 10, group 2). Within 3 months, all patients underwent urodynamic re-evaluation during ongoing tolterodine treatment. RESULTS In group 1, the mean maximum bladder capacity increased from 120.2 to 173.0 mL (+44%), the mean detrusor compliance increased from 8.7 to 13.5 mL/cm H(2)O (+55%), and the mean maximum detrusor pressures decreased from 70.1 to 37.9 cm H(2)O (-46%); the differences were significant (P < 0.001). In group 2, no differences in the urodynamic effects of oxybutynin versus tolterodine were noted. Only 1 patient experienced a transient and moderately adverse effect with tolterodine. CONCLUSIONS Although based on a limited number of subjects, these data indicate that in pediatric patients with detrusor hyperreflexia, tolterodine may be better tolerated than and equally effective as the standard drug oxybutynin chloride.

Basal and Acetylcholine-Stimulated Nitric Oxide Formation Mediates Relaxation of Rabbit Cavernous Smooth Muscle

Externally applied acetylcholine (ACh) in human corpus cavernosum has been shown to cause endothelium-dependent smooth muscle relaxation. Changes in isometric tension in rabbit cavernous smooth muscle strips mounted in organ bath chambers were monitored in the presence of blocking agents. Nitric oxide (NO) is known as an endothelium-derived relaxation factor (EDRF). Addition of specific inhibitors of nitric oxide synthesis, such as L-n-monomethyl arginine (L-NMMA) at 5 x 10(-4) mol/l.. or L-n-nitro arginine (L-NOARG) at 2 x 10(-4) mol/l. to strips precontracted with phenylephrine (PE) at 3.16 x 10(-6) mol/l. led to significant increases in tension. In the presence of L-NMMA or L-NOARG, relaxing effects of ACh at 10(-8)-3.16 x 10(-5) mol/l. mediated by muscarinic receptors were almost completely abolished. These data indicate that rabbit cavernous smooth muscle is under the control of basal NO release. They constitute strong evidence that cholinergically induced endothelial formation of NO plays a crucial role in relaxing cavernous smooth muscle.

Is routine excretory urography necessary at first diagnosis of bladder cancer

PURPOSE We determined whether routine excretory urography (IVP) at initial diagnosis of bladder cancer is useful in screening the upper urinary tract for clinically inapparent urothelial tumors. MATERIALS AND METHODS IVPs and ultrasound findings of 314 patients with bladder cancer were reviewed retrospectively. RESULTS Only 1 silent upper urinary tract tumor was detected with IVP (0.3%), resulting in nephroureterectomy. IVP had no further therapeutic consequences except for destruction of an asymptomatic prevesical stone. IVP was followed by ureterorenoscopy in 5 patients with negative results. Upper urinary tract obstruction could be documented equally well by sonography in all cases. CONCLUSIONS Routine IVP at first diagnosis of bladder cancer is unnecessary.

Nitric oxide mediates relaxation in rabbit and human corpus cavernosum smooth muscle

SummaryWe investigated in vitro the relaxant effect of exogenous acetylcholine (ACh) and electric-field stimulation (EFS) on rabbit and human corpus cavernosum smooth muscle strips (CC) precontracted with phenylephrine. The effects of EFS and ACh were monitored alone, after muscarinic receptor blockade and after inhibition of nitric oxide (NO) formation with l-N-nitroarginine (l-NOARG). In rabbit und human CC, both atropine and l-NOARG abolished the relaxant effects of ACh. The relaxant effects of EFS, however, were only slightly reduced by atropine to 97.5±17.5% in human CC and to 89.0±6.1% in rabbit CC. l-NOARG further reduced the EFS effects to 0.8±1.7% in human CC and to 16.2±8.7% in rabbit CC. In strips obtained from impotent patients with diabetes mellitus, the relaxant effects appeared to be significantly less than in strips from nondiabetic impotent men. Tetrodotoxin blocked the relaxant EFS effects in human and rabbit strips completely. The data indicate the important role of NO in cholinergically induced relaxation of cavernous smooth muscle in rabbits and humans. Our findings support the idea of NO as the nonadrenergic noncholinergic neurotransmitter in penile erection in both species. Rabbit erectile tissue might serve as an in vitro animal model for further investigation.

Urodynamic Effects of Oral Oxybutynin Chloride in Children With Myelomeningocele and Detrusor Hyperreflexia

OBJECTIVES To investigate the effects of oral oxybutynin chloride (OC) on standard urodynamic measures in children with myelomeningocele (MMC) and detrusor hyperreflexia. METHODS Forty-one MMC children with detrusor hyperreflexia (19 boys and 22 girls, aged 2 months to 15 years; mean 4.9 years) were evaluated urodynamically before and within 3 months after initiation of oral OC therapy (0.2 to 0.3 mg/kg/day). Therapy with oral OC was always combined with clean intermittent catheterization (CIC). RESULTS Oral OC treatment caused an increase in bladder capacity from 141 +/- 96 to 197 +/- 99 mL (+ 40%; P < 0.01), a decrease in detrusor pressure at maximal capacity from 45 +/- 32 to 28 +/- 23 cm H2O (-38%; P < 0.01), and an increase in detrusor compliance from 6.5 +/- 5.6 to 16.8 +/- 13.7 mL/cm H2O (+ 158%; P < 0.01). Improvement in urodynamic measures and continence were correlated. After a follow-up of at least 2 years, effective protection of renal function was achieved in 38 of the 41 children (93%) with conservative therapy alone. Adverse effects resulted in discontinuation of oral OC treatment in only 2 cases. CONCLUSIONS Treatment with oral OC and CIC is effective and safe in children with MMC and detrusor hyperreflexia and should be initiated early when indicated by urodynamic findings.

Detection of prostate-specific antigen RNA before and after radical retropubic prostatectomy and transurethral resection of the prostate using “Light-Cycler”-based quantitative real-time polymerase chain reaction

OBJECTIVES To report our initial experience gained in establishing real-time reverse transcriptase-polymerase chain reaction (RT-PCR) detection of prostate-specific antigen (PSA) mRNA using the quantitative online PCR system LightCycler. Many studies have thus far failed to provide the desired proof that the detection of circulating PSA-expressing tumor cells with RT-PCR in the blood samples of patients with prostate cancer (PCa) is a highly sensitive prognostic and course marker. One of the possible reasons is the lack of reliable quantification methods. METHODS Blood samples before and after surgery were obtained from 87 patients who underwent radical prostatectomy for locally confined PCa and 27 patients who underwent transurethral resection of the prostate for benign prostatic hyperplasia. Eight days postoperatively, additional blood samples were obtained from the patients with PCa. Quantitative no-nested RT-PCR for PSA mRNA (291 bp) was performed using the LightCycler system applying the SYBR Green protocol. The number of circulating LNCaP tumor cell-equivalents per sample was estimated from the mean amplification value measured in a given number of LNCaP cells. RESULTS PSA mRNA was detected preoperatively in 19 patients with Stage pT2 tumor (40%) and in 28 patients with tumor greater than Stage pT2 (72%), but in only 2 patients with benign prostatic hyperplasia (8%; analysis of variance, P <0.001). Significant quantitative differences were observed among Stage pT2 disease (1034 LNCaP tumor cell-equivalents/mL), greater than Stage pT2 disease (7830 cells/mL), and benign prostatic hyperplasia (58 cells/mL; analysis of variance for all groups, P <0.001). The correlation between the detection of PSA expression by RT-PCR and the Gleason score and serum PSA value was statistically significant. CONCLUSIONS Our results show that the initial experience with the LightCycler system for PSA-assisted detection of circulating PSA mRNA in PCa by RT-PCR may be a promising preoperative prognostic marker for organ-confined or locally advanced PCa. Long-term follow-up of these patients with PCa must demonstrate the clinical value of molecular diagnostics with quantitative RT-PCR systems.

NITRIC OXIDE MEDIATES NEUROGENIC RELAXATION INDUCED IN RABBIT CAVERNOUS SMOOTH MUSCLE BY ELECTRIC FIELD STIMULATION

We investigated the relaxant effect of electric field stimulation (EFS) on rabbit cavernous smooth muscle strips in vitro precontracted by phenylephrine. Effects of EFS were monitored alone, and following muscarinic receptor blockade, and inhibition of nitric oxide (NO) formation by L-N-monomethylarginine (L-NMMA) or by L-N-nitroarginine (L-NOARG). Atropine only slightly reduced the relaxant effect of EFS to 89.0 +/- 6.1 percent. Additional application of L-NMMA further reduced the relaxant effect to 37.3 +/- 15.3 percent. Substitution of L-NOARG for L-NMMA led to a more pronounced inhibition of relaxant effects to 16.2 +/- 8.7 percent. The results indicate that neurogenically induced relaxation of rabbit cavernous smooth muscle is mediated mainly by NO formation and argue against a substantial role of relaxing peptidergic neurotransmitters, such as vasoactive intestinal polypeptide and calcitonin-gene-related peptide, in penile erection.