Adjuvant vs. progression-triggered treatment with gemcitabine in platinum-ineligible high-risk bladder cancer patients: Long-term follow-up of a randomized phase 3 trial.

BACKGROUND: Cisplatin-based chemotherapy (ChT) is the preferred perioperative treatment in muscle-invasive urothelial carcinoma of the urinary bladder (UCUB). Nevertheless, a certain number of patients are ineligible for platinum-based ChT. This trial compared immediate adjuvant vs. delayed gemcitabine ChT at progression in platinum-ineligible patients with high-risk UCUB. METHODS: High-risk platinum-ineligible UCUB patients (n = 115) were randomized 1:1 to adjuvant gemcitabine (n = 59) or gemcitabine at progression (n = 56). Overall survival was analyzed. Additionally, we analyzed progression-free survival (PFS), toxicity and quality of life (QoL). RESULTS: After a median follow-up of 3.0 years (inter quartile range [IQR]: 1.3-11.6), adjuvant ChT did not significantly prolong overall survival (OS) (HR: 0.84; 95% CI: 0.57-1.24; P = 0.375), with 5-year OS of 44.1% (95% CI: 31.2-56.2) and 30.4% (95% CI: 19.0-42.5), respectively. We noted no significant difference in PFS (HR: 0.76; 95% CI: 0.49-1.18; P = 0.218), with 5-year PFS of 36.2% (95% CI: 22.8-49.7) in the adjuvant group and 22.2% (95% CI: 11.5%-35.1%) when treated at progression. Patients with adjuvant treatment showed a significantly worse QoL. The trial was prematurely closed after recruitment of 115 of the planned 178 patients. CONCLUSIONS: There was no statistically significant difference in terms of OS and PFS for patients with platinum-ineligible high-risk UCUB receiving adjuvant gemcitabine compared to patients treated at progression. These findings underline the importance of implementing and developing new perioperative treatments for platinum-ineligible UCUB patients.

A testosterone-producing Leydig cell tumor metastasis during hormonal treatment of prostate cancer.

We describe a patient with a testosterone-producing metastasis discovered during the follow-up of prostate cancer. The patient had a history of a Leydig cell tumor (LCT) in the right testicle for which he underwent radical orchiectomy at the age of 60 years. Within a year after orchiectomy, he was diagnosed with prostate cancer. He received a radical prostatectomy with pelvic lymph node dissection. Due to recurrent prostate cancer, he underwent salvage radiation to the prostatic fossa and pelvic lymph node stations with hormonal treatment for 3 years. After approximately 1.5 years of chemical castration, a significant increase in testosterone level occurred. Further, diagnostic evaluations and surgery revealed a testosterone-producing LCT metastasis in the retroperitoneum.

Renal cell carcinoma and oxidative stress: The lack of peroxisomes.

Oxidative stress plays an important role in carcinogenesis because of induction of DNA damage and its effects on intracellular signal transduction pathways. Here, we investigated the relationship between the defence against oxidative stress and human renal cell carcinoma that originates from proximal tubular epithelium. Oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase (G6PD) activity is a diagnostic tool for the detection of carcinomas. Its mechanism is based on high G6PD activity, reduced superoxide dismutase activity and reduced numbers of peroxisomes in the cancer cells. Five out of the 8 renal carcinomas studied here demonstrated oxygen insensitivity. These carcinomas showed high G6PD activity, whereas the other 3 carcinomas contained lower G6PD activity and were oxygen sensitive like non-cancer cells. Oxygen insensitivity did not correlate with tumour grade, staging or presence of metastases. Electron microscopy and immunofluorescence of catalase showed large numbers of peroxisomes in epithelial cells of proximal tubules of normal human kidney, whereas these organelles were completely absent in cancer cells of all carcinomas. As a consequence of the absence of peroxisomes in cancer cells, fatty acid metabolism is disturbed in addition to the altered glucose metabolism that is generally observed in cancer cells. Therefore, therapeutic approaches should focus on metabolism in addition to other strategies targeting signal transduction and angiogenesis.

Identification of disease biomarkers by profiling of serum proteins using SELDI-TOF mass spectrometry.

Proteins are the main actors in all physiological and pathological processes. Since the final structure of the protein does not depend on the DNA sequence or even the mRNA sequence alone, the search for direct approaches on the proteome has gained great interest. The most complex and probably the largest proteome is serum, making it clinically the most important. ProteinChip technology, in combination with modern mass spectrometry, allows the complex search for biomarkers, molecular interactions, signaling pathways, and the identification of novel therapeutic compounds. Here we describe the surface-enhanced laser desorption-ionization (SELDI) in combination with the time-of-flight (TOF) mass spectrometry for analyzing serum samples (SELDI was a patented technique from Ciphergen, Fremont, CA). Aluminum-based arrays contain chemical or biological surfaces allowing the capture of proteins, which interact with the surface. The bound proteins are laser desorbed and ionized for mass spectroscopy analysis. The differential mass spectral patterns reflect the protein expression bound on the chip surface and allow the comparison between various samples. Proteins of interest can be identified using peptide mass fingerprinting (PME).

Metastasis is promoted by a bioenergetic switch: new targets for progressive renal cell cancer.

Targeted therapies have demonstrated clinical benefit with limited impact on long-term disease specific survival in the treatment of renal cell cancer (RCC). New opportunities for the treatment of tumors that are resistant or have relapsed, are needed. Increased anaerobic glucose fermentation to lactate (aerobic glycolysis), leading to oxygen- and mitochondria-independent ATP generation is a hallmark of aggressive cancer growth. This metabolic shift results in increased lactate production via cycling through the pentose phosphate pathway (PPP), and plays an important role in tumor immune escape, progression and resistance to immune-, radiation- and chemo-therapy. This study explored the activity and impact of the oxidative and nonoxidative branches of the PPP on RCC to evaluate new therapeutic options. Activity was determined in the oxidative branch by glucose-6-phosphate-dehydrogenase (G6PD) activity, and in the nonoxidative branch by the total transketolase activity and the specific expression of the transketolase-like-1 (TKTL1) protein. Transketolase and G6PD activity were intensely elevated in tumor tissues. Transketolase, but not G6PD activity, was more elevated in metastasizing tumors and TKTL1 protein was significantly overexpressed in progressing tumors (p = 0.03). Lethal tumors, where surrogate parameters such as grading and staging had failed to predict progression, showed intensive TKTL1 protein expression. RCC was found to have activated oxidative and nonoxidative glucose metabolism through the PPP, displaying a bioenergetic shift toward nonoxidative glucose fermentation in progressing tumors. The coexistence of cancer cells with differentially regulated energy supplies provides new insights in carcinogenesis and novel anticancer targets.

Relaxation of human detrusor muscle by selective beta-2 and beta-3 agonists and endogenous catecholamines.

OBJECTIVES: The goal of this study was to identify potent relaxant agents of the human detrusor muscle. Therefore, the relaxant effects of different selective beta (beta)-adrenoceptor agonists were examined. Also, the relaxant effects of the endogenous catecholamines were investigated to functionally characterize the beta-adrenoceptor subtype mainly responsible for adrenergic-mediated relaxation in the detrusor muscle of humans. METHODS: Experiments were performed on muscle strips of human detrusor suspended in a tissue bath. The tissue originated from patients who had undergone total cystectomy. The selective beta3-agonists BRL 37344, ZD 7114, and CGP 12177, the selective beta2-agonists terbutaline and clenbuterol, and the nonselective beta-agonist isoprenaline were investigated. Concentration-relaxation curves of the catecholamines were performed to determine the rank order of potency. RESULTS: The maximal relaxation induced by BRL 37344, ZD 7114, and CGP 12177 was 36%, 39%, and 37%, respectively. The corresponding pD2 values were 6.73, 4.82, and 6.09, respectively. Terbutaline and clenbuterol induced a maximal relaxation of 48% and 27%, and their pD2 value was 4.97 and 5.34, respectively. Isoprenaline, adrenaline, and noradrenaline induced a maximal relaxation of 72%, 58%, and 79%, respectively. The corresponding pD2 values were 6.18, 6.16, and 6.09, respectively. Because their differences were not significant, no rank order of potency was determined. CONCLUSIONS: Beta-adrenergic agonists are potent relaxant agents of the human detrusor muscle in vitro. Both beta2 and beta3-adrenoceptors contribute to adrenergic-mediated relaxation. Our results point to a slightly greater role for the beta3-receptor in human detrusor muscle.

Fluorescence in situ hybridization: a multitarget approach in diagnosis and management of urothelial cancer.

Noninvasive tests for detecting genetic or molecular alterations in urine indicative of urothelial cancer are increasingly becoming the focus of urological cancer research. Since its approval by the US FDA in 2001, the fluorescence in situ hybridization test (Vysis UroVysion) has been widely evaluated. In general, published data demonstrate better sensitivity and equal or better specificity compared with routine cytology, which is still considered the 'gold standard' in diagnosing and monitoring bladder tumors. However, the fluorescence in situ hybridization test seems to provide not only a useful tool in bladder cancer detection, but also in the diagnosis of upper urinary tract tumors, surveillance and determining therapy effectiveness. This multitarget assay that detects four different chromosomal aberrations in tumor cells is a kind of objective molecular cytology and has proven advantages over routinely used cytology.

Inhibitory effects of L- and T-type calcium antagonists on contractions of human detrusor muscle.

The inhibitory and relaxant effects of the L-type calcium antagonists nifedipine, nimodipine, verapamil and diltiazem, and of the T-type calcium antagonist mibefradil, on contractions of isolated human detrusor muscle were investigated. The tissue was obtained from 10 patients undergoing cystectomy due to bladder cancer. Effects of the calcium antagonists at different concentrations on the concentration-response curves for carbachol were investigated. Furthermore, concentration-relaxation curves were performed using potassium-precontracted muscle strips. All L-type calcium antagonists suppressed the mean concentration-response curve of carbachol significantly at a concentration of 10(-6) M. Mibefradil up to 10(-5) M did not significantly suppress it. Nifedipine significantly reduced the carbachol-induced maximum contraction to 75% and 44%, verapamil to 75% and 67% of the appropriate control value at concentrations of 10(-7) and 10(-6) M, respectively. Diltiazem reduced it insignificantly to 96% and 71% at the above-mentioned concentrations. The concentration-relaxation experiments revealed following pD2-values and maximum relaxations of nifedipine, nimodipine, verapamil and diltiazem, respectively: 6.23, 6.37, 5.66, 5.81 and 85%, 83%, 82%, 90%. Maximum relaxations and pD2-values were not significantly different from each other. The lowest concentration, for which a significant effect compared to control in Student;s t-test was found, amounted to 10(-10) M, 10(-9) M, 10(-7) M, 10(-6.5) M and 10(-4) M for nimodipine, nifedipine, diltiazem, verapamil and mibefradil, respectively. L-type calcium antagonists are very potent relaxant agents of the human detrusor muscle in vitro.

La influencia de la "resección transuretral diferenciada" en la recurrencia y progresion en cáncer vesical superficial / The influence of "differentiated transurethral resection"

OBJETIVO: Los datos experimentales muestran correlación entre el área de urotelio dañado y la tasa de recurrencia en cáncer superficial de la vejiga. La adherencia de las células tumorales aumenta frente a la exposición da la matriz extracelular (ECM). La resección transuretral de los tumores vesicales (TURB) descubre el ECM y puede favorecer la recurrencia. La pregunta si la recurrencia aumenta en los casos de grandesáreas de resección es difícil de contestar, porque nunca habrá estudios prospectivos al respecto. En este estudio investigamos si lesiones del urotelio más profundasy más grandes, como las que son causadas por resecciones “diferenciadas” se relacionan con mayores tasas de recurrencia y progresión que resecciones regularesde los tumores vesicales.MÉTODOS: 163 pacientes con cáncer vesical superficialfueron evaluados retrospectivamente. 66 fueron tratadoscon una TURB “diferenciada” y 97 con una TURB regular. Analizamos las tasas de recurrencia y progresiónen un seguimiento mínimo de 48 meses, así como la persistencia de tumor en la re-resección.RESULTADOS: Los pacientes sometidos a TURB “diferenciada”mostraron no tener ninguna incidencia más alta de recurrencia y/o de progresión del tumor, pero mostraron un porcentaje significativamente más alto de resecciones libres de tumor en la re-resección.CONCLUSIONES: Como modelo de lesión urotelial mayor, la técnica de TURB “diferenciada” para cáncer superficial de vejiga no muestra ninguna influencia negativaen las tasas de recurrencia y progresión. Parece improbable que la matriz extracelular descubierta por la resección transuretral sea la razón principal de las altas tasas de recurrencia. Por otro lado, la significativa menor incidencia de persistencia tumoral en la segundaresección favorece a la técnica “diferenciada” de resección en el tratamiento transurethral de los tumores vesicales superficiales

OBJECTIVES: Experimental data show the relationship between the area of injured urothelium and recurrence rates in superficial bladder cancer. Tumor cell adherence is increased by the exposure to the extracellular matrix (ECM). Transurethral resection uncovers the ECM and might lead to recurrent tumors. The question ifrecurrences are increased by enlarged urothelium resection areas is difficult to answer because there will be noprospective studies available. We investigated if deeper and larger urothelial injuries, which are caused by differentiated resections lead to higher recurrence and progression rates than regular resections of bladdertumors.METHODS:163 patients with superficial bladder cancer were retrospectively evaluated. Sixty-six received a differentiated resection and 97 a regular one. We analyzed the recurrence and progression rates over a minimum of 48 months as well as tumor persistence in the second resection.RESULTS: Patients with differentiated resections of bladder tumors were found to have no higher incidence of tumor recurrence and progression but showed a significantly higher percentage of tumor free second resections.CONCLUSIONS: As a model of enlarged urotheliallesions the differentiated resection technique for superficial bladder cancer has no negative influence on recurrence or progression rates. It seems unlikely that the extracellular matrix uncovered by resection is the main reason for the high recurrence rates. The significantly lower incidenceof tumor persistence in the second resection favors the differentiated resection technique in the transurethral treatment

Inhibitory effects of various L-type and T-type calcium antagonists on electrically generated, potassium-induced and carbachol-induced contractions of porcine detrusor muscle.

The inhibitory effects of different calcium antagonists on contractions of isolated porcine detrusor muscle were investigated. Suppression of the maximum potassium-induced contraction and electrically generated contractions by nifedipine, verapamil and diltiazem were investigated. Furthermore, concentration-response curves of carbachol after pretreatment with the L-type antagonists nifedipine, verapamil, diltiazem, nimodipine and the T-type antagonist mibefradil at different concentrations were performed. Nifedipine significantly reduced the potassium-induced maximum contraction to 89, 60, 21, 8 and 4% (10(-9)-10(-5) M). Verapamil and diltiazem significantly reduced it to 64, 30 and 5% (10(-7)-10(-5) M) or 79, 27, 7 and 1% (10(-7)-10(-4) M), respectively. Nifedipine, verapamil and diltiazem significantly reduced the electrically generated contraction to 55, 36, 34 and 25% (10(-7)-10(-4) M), 71, 32 and 2% (10(-6)-10(-4) M), 96, 78, 38 and 5% (10(-7)-10(-4) M), respectively. pD2 values of nifedipine, verapamil and diltiazem amounted to 7.07, 5.56 and 5.40 and differed significantly. After pretreatment with nifedipine at 10(-6) M, the concentration-response curve of carbachol was nearly suppressed. The effects of nimodipine, verapamil and diltiazem were smaller. Mibefradil caused only at 10(-5) M a significant reduction. All investigated L-type calcium antagonists were strong inhibitors of the examined contractions. Nifedipine showed the biggest inhibitory effect.

Adjuvant cisplatin plus methotrexate versus methotrexate, vinblastine, epirubicin, and cisplatin in locally advanced bladder cancer: results of a randomized, multicenter, phase III trial (AUO-AB 05/95).

PURPOSE: Radical cystectomy as standard treatment of muscle-invasive urothelial carcinoma of the urinary bladder cures less than 50% of patients with locally advanced bladder cancer. We compared two adjuvant combination chemotherapies in patients with stage pT3a-4a and/or pathologic node-positive transitional-cell carcinoma of the bladder after radical cystectomy. PATIENTS AND METHODS: A total of 327 patients were randomly assigned to either adjuvant systemic chemotherapy with three cycles of cisplatin 70 mg/qm(2) on day 1 and methotrexate 40 mg/qm(2) on days 8 and 15 of a 21-day cycle (CM) or three cycles of methotrexate 30 mg/qm(2) on days 1, 15, and 22, vinblastine 3 mg/qm(2) on days 2, 15, and 22, epirubicin 45 mg/qm(2) on day 2, and cisplatin 70 mg/qm(2) on day 2 of a 28-day cycle (M-VEC). RESULTS: The hazard ratio for progression-free survival as the primary end point was 1.13 (90% CI, 0.86 to 1.48) for 163 CM patients compared with 164 M-VEC patients whose right-hand limit remained below the upper bound compatible with the noninferiority hypothesis (alpha = .0403). The 5-year progression-free, tumor-specific, and overall survival rates (point estimates +/- SE) for CM versus M-VEC were 46.3% +/- 4.6% v 48.8% +/- 4.5%, 52.0% +/- 4.6% v 52.3% +/- 4.8%, and 46.1% +/- 4.3% v 45.1% +/- 4.6%, respectively. WHO grade 3 and 4 leukopenia occurred in 7.0% of patients treated with CM and 22.2% of patients treated with M-VEC (P < .0001). CONCLUSION: CM cannot be considered inferior to M-VEC with regard to progression-free survival of patients with locally advanced bladder cancer after radical cystectomy. Moreover, patients receiving adjuvant CM combination therapy experienced significantly less grade 3 and 4 leukopenia than patients treated with M-VEC.

Impact of holmium:YAG and neodymium:YAG lasers on the efficacy of DNA delivery in transitional cell carcinoma.

New approaches in the treatment of transitional cell carcinoma (TCC) are using gene therapy to influence the disease at the genetic level. Technical advances in genomics, the availability of tissue-specific gene promoters and other developments have made this approach more realistic. Transporting the gene into the target cell is still the major problem. Several transfection techniques have been introduced. Transfection of naked DNA is one of the simplest to perform but transfection rates have been very poor. We investigated the influence of laser energy on transfection efficacy in urothelial cancer cells in vitro with two types of medical lasers. A suspension of human transitional cancer cells (UM-UC3; 3.5 million cells/ml) was mixed with 200 microg of plasmid DNA (pEGFP-N1). Two types of laser energy, neodymium:YAG (Nd:YAG) and holmium:YAG (Ho:YAG), were applied to the cell suspension in different energy settings. Twenty four hours after treatment, transfection rates were measured with FACS analysis. Energy setting parameters that determine the efficacy of laser were investigated. The significance of different transfection rates was estimated with the student's t-test. We demonstrated that the Nd:YAG laser was not suitable for achieving significant transfection of the reporter gene to the cells. In contrast, the Ho:YAG laser produced satisfactory transfection rates. There was an increase in transfection with increasing frequency of laser pulses, from 16% with 2 Hz up to 40% with 10 Hz (p < 0.0005). Pulse frequency was therefore stabilised at 10 Hz. Pulse energy (mJ) showed the same dependency: a transfection rate of 18.3% was achieved with 1,000 mJ and 53.8% with 2,000 mJ (p > 0.0005). Additionally, we investigated the impact of total pulse number (imp) with different pulse energies. At 1,000 mJ, a transfection rate of 18.3% was estimated with 200 imp and 48.56% with 750 imp, (p < 0.0005). At 2,000 mJ, a transfection rate of 53.8% was achieved with 200 imp and 58.26% with 500 imp. The optimal laser setting observed in this experiment was 10 Hz, 2,000 mJ and 500 imp. This study indicates that the efficacy of naked DNA delivery into TCC in vitro is improvable by application of Ho:YAG laser energy. The Nd:YAG laser did not increase transfection rates in our model. Our results with the Ho:YAG laser are encouraging for further studies to optimise DNA delivery. As TCC tissue is relatively easy to access, this method could become an effective and minimally invasive procedure in urothelial cancer treatment.

Cytotoxicity of cisplatin in bladder cancer is significantly enhanced by application of bcl-2 antisense oligonucleotides.

BACKGROUND: The aim of our study was to examine the effects of the combined application of cisplatin and bcl-2 antisense oligonucleotide on human bladder cancer cell lines to determine the possible synergistic effects in cytotoxicity and to estimate its potential value for subsequent in vivo trials. MATERIALS AND METHODS: Human bladder cancer cell lines (UM-UC 3, RT 112, T24/83 and HT 1197) were treated with bcl-2 antisense oligonucleotide, cisplatin, or a combination of both and incubated for 48 h under standard conditions. Cell survival was determined using a Neubauer haemocytometer or standard MTT assay. BCL-2 expression was verified using western blotting. RESULTS: The combined treatment resulted in significant lower cell survival rates compared to individual treatment. Additionally, there was a decrease in cell survival rate with an increase in cisplatin concentration in combined treatment that was not observed in cisplatin mono treatment. CONCLUSIONS: For the combined treatment with oligonucleotides and cisplatin a synergistic effect can be strongly suggested. Therefore, further investigations and in vivo trials have to be done to determine the possible benefits for clinical applications.

High-intensity focused ultrasound for ex vivo kidney tissue ablation: influence of generator power and pulse duration.

BACKGROUND AND PURPOSE: The therapeutic application of noninvasive tissue ablation by high-intensity focused ultrasound (HIFU) requires precise physical definition of the focal size and determination of control parameters. The objective of this study was to measure the extent of ex-vivo porcine kidney tissue ablation at variable generator parameters and to identify parameters to control lesion size. MATERIALS AND METHODS: The ultrasound waves generated by a cylindrical piezoceramic element (1.04 MHz) were focused at a depth of 100 mm using a parabolic reflector (diameter 100 mm). A needle hydrophone was used to measure the field distribution of the sound pressure. The morphology and extent of tissue necrosis were examined at generator powers of up to 400 W (P(el)) and single pulse durations of as long as 8 seconds. RESULTS: The two-dimensional field distribution resulted in an approximately ellipsoidal focus of 32 x 4 mm (-6 dB). A sharp demarcation between coagulation necrosis and intact tissue was observed. Lesion size was controlled by both the variation of generator power and the pulse duration. At a constant pulse duration of 2 seconds, a generator power of 100 W remained below the threshold doses for inducing a reproducible lesion. An increase in power to as high as 400 W induced lesions with average dimensions of as much as 11.2 x 3 mm. At constant total energy (generator power x pulse duration), lesion size increased at higher generator power. CONCLUSIONS: This ultrasound generator can induce defined and reproducible necrosis in ex-vivo kidney tissue. Lesion size can be controlled by adjusting the generator power and pulse duration. Generator power, in particular, turned out to be a suitable control parameter for obtaining a lesion of a defined size.

Alteration of the LRP1B gene region is associated with high grade of urothelial cancer.

We have delineated regions of interest at chromosome 2q21.2, 2q36.3, and 2q37.1 by deletion mapping of 114 urothelial cancers (UC). Altogether, 17%, 18%, and 63% of the G1, G2, and G3 tumors displayed loss of heterozygosity at chromosome 2q, respectively, The region at 2q21.2 was narrowed down to the LRP1B gene (NT_005129.6). Hemi- and homozygous deletion at the LRP1B gene region was seen in 31 of 114 UCs. Only 8% of the UCs with G1 and none with G2 tumors showed loss of heterozygosity at the LRP1B gene, whereas 49% of the G3 UCs had allelic loss at this region. RT-PCR analysis of the LRP1B gene showed the lack of expression of several exons in 2 of 9 cases analyzed. Our analysis suggests that the LRP1B gene is a candidate tumor suppressor gene in UCs.