Subclassification, survival prediction and drug target analyses of chemotherapy-naïve muscle-invasive bladder cancer with a molecular screening.

BACKGROUND: Transcriptome expression studies identified distinct muscle invasive bladder cancer (MIBC) subtypes closely related with breast cancer subclasses. Here we developed a sensitive quantification method for MIBC subclassification (luminal, basal, p53-like). In addition, the subtype specific expression of drug targets has been investigated. METHODS: Absolute quantification (nCounter) of a 64-gene panel was performed on MIBC patients (n=47) treated exclusively with radical cystectomy (RC). In conjunction of 170 MIBCs from 3 independent cohorts, a minimal set of consensus genes has been established. Survival of the consensus subtypes has been assessed by multivariate analysis. Relevant drug targets were tested for their subtype specificity in a clustering independent assessment. RESULTS: A reduced 36-gene panel stably clustered into 3 subtypes throughout the cohorts (luminal, basal, infiltrated). Patients treated by RC only, showed worst 8-year disease specific survival (DSS) for the luminal subtype in contrast to the infiltrated subtype (17% vs. 73%, p=0.011). In multivariate analyses, the risk stratification based on luminal versus not-luminal MIBC proved to be an independent predictor for DSS superior to the TNM system in patients with RC. Drug targets (e.g. ERBB2, FGFR, AR, PDGFRB) showed a distinct subtype attribution. The subtypes based on this nCounter screening could further be validated by the TCGA cohort. CONCLUSION: This MIBC subtype screening predicted survival and allowed an analysis of subtype specific drug targets, thus being a powerful tool for the translation of personalized MIBC treatment concepts.

Decreased Invasion of Urothelial Carcinoma of the Bladder by Inhibition of Matrix-Metalloproteinase 7.

OBJECTIVES: To measure and to modulate the invasive potential of urothelial carcinoma of the bladder (UCB) cells in a standardized preclinical setting using broad-spectrum matrix-metalloproteinase (MMPs) inhibitors and specific targeting of MMP7. MATERIALS AND METHODS: MMP expression levels in UCB cells were determined by quantitative real-time PCR (qRT-PCR) and gel zymographies of cell supernatants (MMP9, MMP2 and MMP1) and cell lysates (MMP7). The invasiveness of human UCB cells (HT1197 and T24/83) and human benign urothelial cells (UROtsa) was modulated by a broad-spectrum MMP inhibitor (4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic acid; AHA) and by MMP7 specific siRNAs. MMP7 knockdown efficiency was assessed by qRT-PCR and western blot. Invasive potential of UCB cells was measured by a standardized trans-epithelial electrical resistance (TEER) assay. RESULTS: Different MMP secretion profiles were measured in UCB cells. The active form of MMP7 was exclusively detected in HT1197 cells. Characteristic TEER breakdown patterns were observed in UCB cells when compared to benign cells. Invasive potentials were significantly higher in HT1197 cells than in T24/83 and in UROtsa cells [14.8±5.75 vs. 1.5±0.56 and 1.2±0.15, respectively; p < 0.01]. AHA treatment reduced the invasive potential of HT1197 cells. Also the specific downregulation of MMP7 by siRNA lowered the HT1197 cell invasiveness [20±1.0 vs. 16±2.8; p < 0.05]. Neither AHA nor MMP-7 siRNA transfection altered the invasive potential of T24/83 cells. CONCLUSIONS: Invasion of UCB is partially dependent on MMPs. Specific targeting of MMP7 by siRNA reduces the invasive potential in a subgroup of UCB cells. Therefore, MMP7 represents a potential therapeutic target which warrants further investigation.

Impact of Altered WNT2B Expression on Bladder Wall Fibroblasts: Implications for Apoptosis Regulation in the Stroma of the Lower Urinary Tract.

BACKGROUND: Little is known about the role of WNT signalling in pathological processes involving the urinary tract stroma. Here the impact of WNT signalling on bladder wall fibroblasts (BWFs) was studied using integrated expression profiling. MATERIAL AND METHODS: WNT ligand and downstream WNT pathway component expression was profiled in human BWFs using qRT-PCR. Highly expressed WNT2B was knocked down using siRNA in BWFs. The expression of 730 mRNAs and 800 miRNAs was analyzed on the nCounter MAX platform in #WNT2B and control transfected BWFs. qRT-PCR was used for validation in vitro and in matched scar and healthy bladder wall tissue samples of 12 patients with vesico-urethral anastomotic stricture (VUAS). RESULTS: Thirteen genes and 9 miRNAs showed differential expression in #WNT2B cells. Among these were TNFSF10, a key apoptosis inductor, (0.22fold, p = 0.011) and miR-1246 (36.2fold, p = 0.031). miRNA target prediction indicated TNFSF10 to be regulated by miR-1246. qRT-PCR analysis confirmed differential expression of miR-1246 and TNFSF10 in #WNT2B BWFs. Furthermore, TNFSF10 was significantly underexpressed in VUAS tissue (p = 0.009). CONCLUSION: Perturbation of WNT signalling results in an altered expression of the apoptosis inductor TNFSF10. Similar changes are observed in VUAS. Further studies investigating the crosslink between WNT signalling and apoptosis regulation in the urinary tract stroma are warranted.

The EEF1A2 gene expression as risk predictor in localized prostate cancer.

BACKGROUND: Besides clinical stage and Gleason score, risk-stratification of prostate cancer in the pretherapeutic setting mainly relies on the serum PSA level. Yet, this is associated with many uncertainties. With regard to therapy decision-making, additional markers are needed to allow an exact risk prediction. Eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) was previously suggested as driver of tumor progression and potential biomarker. In the present study its functional and prognostic relevance in prostate cancer was investigated. METHODS: EEF1A2 expression was analyzed in two cohorts of patients (n = 40 and n = 59) with localized PCa. Additionally data from two large expression dataset (MSKCC, Cell, 2010 with n = 131 localized, n = 19 metastatic PCa and TCGA provisional data, n = 499) of PCa patients were reanalyzed. The expression of EEF1A2 was correlated with histopathology features and biochemical recurrence (BCR). To evaluate the influence of EEF1A2 on proliferation and migration of metastatic PC3 cells, siRNA interference was used. Statistical significance was tested with t-test, Mann-Whitney-test, Pearson correlation and log-rank test. RESULTS: qRT-PCR revealed EEF1A2 to be significantly overexpressed in PCa tissue, with an increase according to tumor stage in one cohort (p = 0.0443). In silico analyses in the MSKCC cohort confirmed the overexpression of EEF1A2 in localized PCa with high Gleason score (p = 0.0142) and in metastatic lesions (p = 0.0038). Patients with EEF1A2 overexpression had a significantly shorter BCR-free survival (p = 0.0028). EEF1A2 expression was not correlated with serum PSA levels. Similar results were seen in the TCGA cohort, where EEF1A2 overexpression only occurred in tumors with Gleason 7 or higher. Patients with elevated EEF1A2 expression had a significantly shorter BCR-free survival (p = 0.043). EEF1A2 knockdown significantly impaired the migration, but not the proliferation of metastatic PC3 cells. CONCLUSION: The overexpression of EEF1A2 is a frequent event in localized PCa and is associated with histopathology features and a shorter biochemical recurrence-free survival. Due to its independence from serum PSA levels, EEF1A2 could serve as valuable biomarker in risk-stratification of localized PCa.

RAB27A, RAB27B and VPS36 are downregulated in advanced prostate cancer and show functional relevance in prostate cancer cells.

Paracrine and long-range signaling via extracellular vesicles, such as exosomes and microvesicles, is deemed crucial for tumorigenesis, invasion and spread of solid tumors. The ESCRT machinery (endosomal sorting complexes required for transport) and Rab-proteins act as key players in vesicular trafficking and secretion. Yet, their role in prostate cancer (PCa) is unknown. Therefore, this study aimed to elucidate the relevance of these components in PCa. In silico reanalysis of genes with known involvement in vesicular trafficking and secretion in an existing microarray dataset revealed low expression of RAB27A, RAB27B and VPS36 to be predictive for reduced BCR-free survival in patients with localized PCa (p=0.033, 0.025 and 0.005). In the same microarray dataset underexpression of RAB27A, RAB27B and VPS36 was seen in distant metastases (p<0.001; p=0.003; p<0.001). This was consistent in two further microarray datasets. qRT-PCR-validation in two independent cohorts of PCa specimens (n=90) showed low expression of VPS36 in PCa tissue (p=0.023), especially in castration-resistant tumors (p=0.002). In all five datasets there were significant correlations between the expression of at least two of the candidates. Upon knockdown of VPS36 an increase of RAB27A and RAB27B expression, but not vice versa, was observed in both prostate and breast cancer cells (PC3, MDA-MB­231). In PC3 cell knockdown of RAB27B and VPS36 dramatically reduced colony formation (-52.2%, p<0.001; -71.1%, p<0.001) and, controversial to reports in other tumor entities, increased the release of extracellular particles (+25.3%, p=0.014; +45.6%, p<0.001). Taken together RAB27A, RAB27B and VPS36 are frequently underexpressed in advanced PCa and are inversely correlated with PCa outcome. There seems to be a close relationship in the expression of RAB27A, RAB27B and VPS36, with RAB27A and RAB27B being dependent on VPS36. Changes in colony formation and particle release upon RNAi indicate an involvement in paracrine cell-cell communication.

Expression of the p53 Inhibitors MDM2 and MDM4 as Outcome Predictor in Muscle-invasive Bladder Cancer.

AIM: To evaluate the prognostic role of the p53-upstream inhibitors MDM2, MDM4 and its splice variant MDM4-S in patients undergoing radical cystectomy (RC) for muscle-invasive bladder cancer (MIBC). MATERIALS AND METHODS: mRNA Expression levels of MDM2, MDM4 and MDM4-S were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) in 75 RC samples. Logistic regression analyses identified predictors of recurrence-free (RFS) and cancer-specific survival (CSS). RESULTS: High expression was found in 42% (MDM2), 27% (MDMD4) and 91% (MDM4-S) of tumor specimens. Increased MDM2 expression was significantly associated with higher tumor stage (p=0.05) and lymphovascular invasion (LVI) (p=0.041). In the univariate analysis, low MDM4 expression (hazard ratio (HR)=5.93; p=0.002; HR=3.00; p=0.047), but not MDM2 (HR=1.63; p=0.222; HR=1.59; p=0.27), were associated with RFS and CSS. In the multivariate analysis, the combination of low MDM4 and high MDM2 was significant for RFS and CSS (HR=14.9; p=0.001; HR=5.63; p=0.019). CONCLUSION: The combination of MDM2 and MDM4 expression is an independent predictor in patients undergoing RC for MIBC.

Expression and predictive value of lymph-specific markers in urothelial carcinoma of the bladder.

OBJECTIVE: To evaluate the expression of multiple lymph-specific markers and to test its association with histopathological characteristics and clinical outcomes in patients with urothelial carcinoma of the bladder (UCB) treated by radical cystectomy (RC). PATIENTS AND METHODS: Vascular endothelial growth factor-C and -D (VEGF-C/-D), its receptor VEGF receptor-3 (VEGFR-3), and chemokine receptor type 7 (CCR7) expressions were assessed by immunohistochemistry in RC specimens of 119 patients. Semiquantitative analyses of marker expressions were correlated with clinical and pathological characteristics. Univariable and multivariable analyses were performed to identify predictors of disease-specific survival (DSS) and recurrence free survival (RFS). RESULTS: VEGF-C, VEGF-D, VEGFR-3, and CCR7 were overexpressed in 37.8%, 26.2%, 50.4%, and 23.5% of UCB samples, respectively. VEGF-D overexpression was significantly associated with a positive lymph node status (pN+). On univariable analysis, a higher pT stage, pN+, the presence of lymphovascular invasion (LVI) and vascular invasion (VI) (all P<0.001), and overexpressions of VEGF-D (P = 0.049) and VEGFR-3 (P = 0.032) were significantly associated with reduced DSS. On multivariable analysis, pT stage (P = 0.002) and pN+status (P = 0.009) were identified as independent predictors of reduced DSS. In a subgroup of patients without lymph node metastasis (pN0; n = 75), pT stage (P = 0.043) and VEGFR-3 overexpression (P = 0.008) were independent predictors of reduced DSS. CONCLUSION: Lymph-specific markers are frequently overexpressed in UCB. VEGF-D overexpression is associated with the presence of lymphatic metastasis. In patients without lymph node metastasis at the time of RC, an assessment of VEGFR-3 expression may improve the identification of high-risk patients. These findings require prospective validation to determine the potential benefit of more aggressive adjuvant treatment.

Assessing the invasive potential of bladder cancer: development and validation of a new preclinical assay.

PURPOSE: We developed and validated an electrophysiological method for standardized preclinical assessment of the invasive potential of urothelial carcinoma of the bladder. MATERIALS AND METHODS: Human UMUC-3, RT-112, HT-1197 and T24/83 bladder urothelial carcinoma cells, and UROtsa benign urothelial cells were co-cultivated with high resistance MDCK-C7 cells seeded below a 0.4 µm pore membrane of an insert to avoid physical contact and cellular migration. Transepithelial electrical resistance in Ω cm(2) across the MDCK-C7 monolayer was measured longitudinally. Invasive potential coefficients were calculated based on the secretion of proteolytic factors by invading cells. RESULTS: Consistent transepithelial electrical resistance breakdown patterns were reproduced in 14 or more independent samples of each cell line. Coefficients of invasive potential were significantly higher in bladder urothelial carcinoma than UROtsa cells, including a mean ± SD of 1.5 ± 0.32 vs 9.9 ± 4.97 in UMUC-3, 12.5 ± 6.61 in T24/83, 20.5 ± 4.24 in RT-112 and 21.0 ± 5.15 in HT-1197 cells (p <0.001). No correlation was found between the secretion patterns of matrix metalloproteinase-1, 2 and 9, and invasive potential. Stimulation of UROtsa cells with recombinant human epidermal growth factor up-regulated matrix metalloproteinase-9 secretion and significantly increased invasive potential a mean of 1.3 ± 0.22 vs 14.6 ± 3.28 after stimulation with 10 ng/ml epidermal growth factor (p <0.001). CONCLUSIONS: We developed a highly sensitive translational tool to study the initial process of metastatic spread of urothelial carcinoma of the bladder. The presented electrophysiological invasion assay enables reliable quantification of the invasive potential of bladder urothelial carcinoma cells before physical transmigration. It can be used to identify key molecules for bladder urothelial carcinoma invasion and develop new therapeutic strategies.

Lymph vessel density in seminomatous testicular cancer assessed with the specific lymphatic endothelium cell markers D2-40 and LYVE-1: correlation with pathologic parameters and clinical outcome.

OBJECTIVES: To evaluate the role of lymph vessel density (LVD) and lymphangiogenesis in seminomatous testicular cancer (STC) by using the lymphatic endothelial cell (LEC) markers LYVE-1 and D2-40. METHODS AND MATERIALS: Paraffin embedded tumor specimens from 40 patients with STC were stained by specific D2-40 and Lyve-1 antibodies. LVD was measured in different representative and standardized areas. Fluorescence double immunostaining for Lyve-1 and Ki-67 was performed and results were correlated with clinicopathologic data. The median follow-up period was 55 (range 10-135) months. RESULTS: Mean intratumoral LVD (D2-40: 1.30 ± 1.99; Lyve-1: 1.82 ± 2.34) was significantly lower than peritumoral LVD (D2-40: 4.94 ± 2.58; Lyve-1: 4.62 ± 2.73) and LVD in nontumoral areas (D2-40: 4.81 ± 3.79; Lyve-1: 4.22 ± 3.19). There was no significant difference between LVD measures when using D2-40 or LYVE-1. Detection rates of lymphatic vascular invasion (LVI) were significantly higher than in conventional HE-stained sections (77.5% vs. 52.5%). No proliferating lymphatic vessels were found. CONCLUSIONS: We found that LVD is decreased within tumor areas of STC. Despite a higher peritumoral LVD, no signs of proliferating endothelial cells were observed, suggesting a lack of lymphangiogenesis in STC. Detection of LVI can be optimized by specific D2-40 or LYVE-1 staining.

HERV-E-mediated modulation of PLA2G4A transcription in urothelial carcinoma.

Human endogenous retroviruses (HERV) and related elements account for more than 8% of the human genome and significantly contribute to the human transcriptome by long terminal repeat (LTR) promoter activity. In this context, HERVs are thought to intervene in the expression of adjacent genes by providing regulatory sequences (cis-effect) or via noncoding RNA including natural antisense transcripts. To address the potential impact of HERV activity in urothelial carcinoma, we comparatively analyzed the HERV transcription profiles in paired samples of non-malignant urothelium and urothelial carcinoma derived from 13 patients with bladder cancer by means of a retrovirus-specific microarray (RetroArray). We established a characteristic HERV signature consisting of six ubiquitously active HERV subgroups (E4-1, HERV-Rb, ERV9, HERV-K-T47D, NMWV3, HERV-KC4). The transcription pattern is largely identical in human urothelial carcinoma, non-malignant urothelial tissue, four tumor-derived cell lines and in a non-malignant urothelial cell line (UROtsa). Quantitative reverse transcriptase PCR (qRT-PCR) of HERV-E4-1, HERV-K(HML-6) and HERV-T(S71-TK1) revealed a bias to lower HERV activity in carcinoma samples compared to non-malignant tissue. Determination of active HERV-E4-1 loci by cloning and sequencing revealed six HERV-E4-1 proviral loci that are differentially regulated in urothelial carcinoma cells and normal tissue. Two full-length HERV-E4-1 proviruses, HERV-Ec1 and HERV-Ec6, are located in antisense orientation in introns of the genes PLA2G4A and RNGTT, respectively. PLA2G4A encodes a cytosolic phospholipase A2 (cPLA2) that is dysregulated in many human tumors. PLA2G4A and HERV-Ec1 displayed reciprocal transcript levels in 7 of 11 urothelial carcinoma patients. Moreover, reciprocal shifts were observed after treatment of UROtsa cells with HERV-Ec1 and PLA2G4A-directed siRNAs or 5-aza-2'-deoxycytidine (aza-dC) pointing to an antagonistic regulation of PLA2G4A and HERV-Ec1 transcription in human urothelial cells. We suggest that transcription of HERV-Ec1 contributes to fine tuning of cPLA2 expression, thereby facilitating tumorigenesis.

New insights into the influence of cigarette smoking on urothelial carcinogenesis: smoking-induced gene expression in tumor-free urothelium might discriminate muscle-invasive from nonmuscle-invasive urothelial bladder cancer.

Smoking is the main risk factor for urothelial bladder cancer. In former smokers the risk decreases but does not reach the low level of never smokers. This indicates reversible and permanent smoking-derived genetic alterations. Transcriptional changes may point to mechanisms, how smoking promotes urothelial bladder cancer. To identify smoking-derived transcriptional changes we performed gene expression profiling in current, former, and never smokers, using tumor and tumor-free urothelium from patients with nonmuscle-invasive urothelial bladder cancer (NMIBC) or muscle-invasive urothelial bladder cancer (MIBC). Smoking turned out to influence gene expression much less than tumor stage (NMIBC or MIBC) and tumor transformation (tumor-free or tumor). Smoking seemed to influence gene expression in patients with MIBC more strongly compared to those with NMIBC. The least irreversible changes after smoking cessation were proposed in tumor-free urothelium from patients with NMIBC. Growth factors and oncogenes were up-regulated in tumor-free urothelium from smokers with MIBC but not from smokers with NMIBC. A panel of genes up-regulated in smokers have potential for early detection and distinction of MIBC from NMIBC using tumor-free tissue.

Smoking increases transcription of human endogenous retroviruses in a newly established in vitro cell model and in normal urothelium.

Human endogenous retroviruses (HERVs) accounting for 9% of the human genome are considered as surrogate markers for genetic instability and as a driving force of genetic variation. Moreover, they modulate regular gene activities and give rise to expression of disease-associated peptides that may serve as diagnostic markers or even targets for T cell-based immune responses. To date, no data are available on the potential link between urothelial carcinogenesis, HERV activity, and tobacco smoking, the main risk for bladder cancer. Here, we report on potential alterations in HERV transcription induced by smoking in a newly established in vitro model and in human urothelium. Normal human dermal fibroblasts were cultivated with urine from never (n = 6) and current smokers (n = 6) and transcription levels for the HERV subfamilies HERV-E 4-1, HERV-T S71-TK1, and HERV-K HML-6 were measured by quantitative real-time PCR. Tendencies toward increased mean transcript levels were detected for cells treated with urine from current smokers. Equally, activity measured in human urothelium supported an increase of HERV transcription in current smokers (n = 9) compared to never smokers (n = 4).

The lymphatic system and its specific growth factor vascular endothelial growth factor C in kidney tissue and in renal cell carcinoma.

OBJECTIVE To determine the role of vascular endothelial growth factor C (VEGF-C) and the quantitative extent of the lymphatic system in renal cell carcinoma (RCC) to analyse a possible correlation with the metastatic spread of cancer cells. PATIENTS AND METHODS In all, 44 patients with clear cell RCC and 12 with papillary or chromophobe RCC were included in an immunohistochemical study. The lymphatic vessel density (LVD) was assessed in the tumour body, the tumour capsule and the tumour-free adherent renal tissue. The expression of VEGF-C was semiquantitatively assessed by the percentage of positive epithelial and cancer cells. Data were analysed for any correlation with the clinicopathological variables. RESULTS The clear and papillary cell RCC contained no lymphatic vessels (0.2, sd 0.8). Chromophobe RCC specimens showed scattered obliterated vessels (0.0-7.7). Several lymphatic vessels were found in the normal renal tissue, mainly associated with blood vessels (2.9, sd 1.9). The highest mean (sd) LVD was in the tumour capsule, of 6.9 (3.4). There was no statistical correlation between the LVD and VEGF-C. In clear cell RCC the expression of VEGF-C was correlated with the size of the tumour (P = 0.042). CONCLUSIONS RCC does not promote the growth of its own lymphatic vessels. The role of a high LVD in the tumour capsule needs to be determined. The VEGF-C staining pattern in normal kidney tissue hints at functions other than lymphangiogenesis.

Lymphangiogenesis occurs in upper tract urothelial carcinoma and correlates with lymphatic tumour dissemination and poor prognosis.

OBJECTIVE: To describe the lymphatic vessel density and to determine the functional and prognostic significance of tumoral lymphatic vessels in upper tract urothelial carcinoma (UTUC). PATIENTS AND METHODS: The study included 65 patients who had a radical nephroureterectomy (RNU) for UTUC between 1997 and 2004. All pathological slides were re-evaluated by one reference pathologist and clinical data were reviewed. Lymphatic endothelial cells (LECs) were stained immunohistochemically using D2-40. The lymphatic vessel density (LVD) was described in representative intratumoral (ITLVD), peritumoral (PTLVD) and non-tumoral (NTLVD) areas. Random samples were selected for double-immunostaining with D2-40 and CD-34 (to distinguish blood and lymphatic vessels) and the proliferation marker Ki-67 to detect lymphangiogenesis. The primary outcome measures were disease-specific survival (DSS) and disease recurrence (urothelial and/or distant). RESULTS: The median (interquartile range) PTLVD was 4.0 (3.0-6.3), and significantly higher than that for ITLVD, of 0.3 (0-1.7) (P < 0.001), and NTLVD, of 3 (2.0-3.7) (P < 0.001). Both a higher ITLVD and PTLVD, the presence of lymphovascular invasion (LVI) (each P < 0.001) and a high tumour grade (P = 0.004) were associated with reduced DSS on univariate analysis. A higher PTLVD (P = 0.028) and the presence of LVI (P = 0.020) independently predicted reduced DSS on multivariate analysis. IT and PT lymphatic vessels showed proliferating LECs in all analysed samples. CONCLUSION: Lymphangiogenesis is present in UTUC, as shown by a significantly increased PTLVD and proliferating LECs. Our findings suggest functional relevance of PT lymphatic vessels during lymphatic tumour spread. PTLVD is a potential novel prognostic factor for DSS in UTUC, and further prospective studies will be needed to determine the effect of its routine evaluation on clinical outcomes of this malignancy.

Topical chemotherapy in human urothelial carcinoma explants: a novel translational tool for preclinical evaluation of experimental intravesical therapies.

BACKGROUND: Urothelial carcinoma (UC) is associated with a high local recurrence rate despite intravesical therapy. There is a lack of representative preclinical models for standardized testing of novel experimental therapies. OBJECTIVE: To develop an ex vivo model for human UC and to evaluate its ability to generate reproducible and reliable results when testing cytotoxic agents. DESIGN, SETTING, AND PARTICIPANTS: Normal human urothelium (NHU) and bladder UC explants were collected from patients treated at our institution. A total of 195 surgical explants were cultured on a gelatine matrix. Tissue viability was regularly assessed using nicotinamide adenine dinucleotide (NADH) diaphorase enzymehistochemistry. Topical paclitaxel (PTX) or mitomycin C (MMC) chemotherapy was performed in a subset of 45 UC specimens. INTERVENTION: All patients underwent radical cystectomy (RC) or primary transurethral resection (TUR) of a bladder UC. MEASUREMENTS: Triple immunofluorescence (pan-cytokeratin [pan-CK]; 4',6-diamidin-2'-phenylindol-dihydrochloride [DAPI]; terminal deoxynucleotidyl transferase biotin-dUTP nick-end labelling [TUNEL]) and caspase-3 staining of paraffin sections was performed. Proliferation rates were assessed using Ki-67 labelling indices. Apoptosis (percent) was quantified in representative tissue areas to characterize culture stability and to assess antineoplastic effects. RESULTS AND LIMITATIONS: No signs of necrosis and no significant changes in apoptosis were observed during the first 12 d of culture. Of all explants, 88.5% were vital after 20 d. In a highly reproducible fashion, topical chemotherapy resulted in significantly increased apoptosis (37.4% [19.0-75.0%] for PTX and 36.2% [18.8-46.7%] for MMC) compared with controls (7.5% [3.0-26.8%]; p<0.001]). No statistically significant difference was observed regarding the effects of the two chemotherapeutic agents (p=0.119). CONCLUSIONS: The presented human ex vivo model takes UC heterogeneity into account and serves as a valuable translational tool. It offers an attractive alternative to preclinical cell line experiments or animal models and may even be used for prospective toxicity and drug efficacy tests in individual patients.

In vivo evaluation of intravesical paclitaxel and combined bcl-xL antisense oligodeoxynucleotide treatment for orthotopic urothelial carcinoma.

PURPOSE: To evaluate intravesical paclitaxel monotherapy and combined treatment with antiapoptotic bcl-xL antisense oligodeoxynucleotides (AS-ODNs) on urothelial carcinoma (UC). METHODS: Forty-eight FoxN(rnu) athymic nude rats with orthotopic human bladder UC were randomized to four treatment groups [1, paclitaxel; 2, paclitaxel/bcl-xL AS-ODNs; 3, bcl-xL AS-ODNs (control); 4, medium (control)]. Three consecutive instillations were applied and weekly endoscopic tumor size measurements were performed. RESULTS: Significant tumor size reduction was achieved in groups 1 and 2 (each P < 0.0001), whereas continuous UC growth was observed in control animals (groups 3 and 4; P < 0.0001 and P < 0.0020). Complete tumor eradication was achieved in four treated animals (groups 1 and 2). No significant difference in chemoresection effects was found between groups 1 and 2 (P = 0.2251). CONCLUSIONS: We present an in vivo evaluation of intravesical treatment with paclitaxel and combined bcl-xL AS-ODNs. Despite efficient tumor size reduction, no gain was observed when adding bcl-xL AS-ODNs in this experimental setting.

Evaluation of cytotoxic effects induced by bcl-2 and bcl-xL antisense-oligodeoxynucleotides in normal urothelium and transitional cell carcinoma.

We report on cytotoxic effects of bcl-2 and bcl-xL antisense-oligodeoxynucleotides (AS-ODNs) in benign urothelial and transitional cell carcinoma (TCC) cell lines. The benign urothelial cell line (UROtsa) and four TCC cell lines (UM-UC-3, RT 112 , HT 1197 and T 24/83) were incubated with bcl-2 and bcl-xL AS-ODNs and cell mortality rates were assessed. Bcl-2 and bcl-xL AS-ODN treatment resulted in low toxicity in UROtsa cells (6% and 10% cell mortality, respectively). After bcl-2 AS-ODN treatment, cell mortality rates in TCC cell lines were significantly higher than in UROtsa cells (mean values: 33% vs. 6%, respectively). Bcl-2 AS-ODN treatment also caused significantly higher cell death rates in the majority of TCC cell lines when compared to bcl-xL-AS-ODN therapy (mean values: 33% vs. 11%, respectively). In conclusion, bcl-2 AS-ODNs show significantly higher cell mortality in TCC cells, whereas toxic effects on normal urothelium seem to be minor. Our results suggest favourable characteristics for the clinical application of AS-ODN in intravesical chemotherapy of TCC.

Serum vascular endothelial growth factor C level in patients with prostate cancer and benign prostatic hyperplasia.

OBJECTIVE: To compare serum vascular endothelial growth factor C (VEGF-C) levels in patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa) and analyze VEGF-C levels in relation to clinicopathologic parameters. STUDY DESIGN: Fifty-eight patients with PCa and 61 patients with BPH were included in this study. Serum VEGF-C levels were assessed by enzyme-linked immunosorbent assay. RESULTS: The serum VEGF-C level for patients with PCa was 3,432.06 +/- 1,851.07 as opposed to 3,166.68 +/- 1,921.2 for patients with BPH. There was no statistically significant difference between the 2 groups (p = 0.4448). There was no correlation of VEGF-C to tumor stage, grading or the preoperative prostate-specific antigen values. CONCLUSION: We cannot recommend VEGF-C serum level as a marker for tumor growth in PCa.

Prognostic implications of lymphangiogenesis in muscle-invasive transitional cell carcinoma of the bladder.

OBJECTIVES: Aim of the study was to describe and evaluate the association of lymph vessel density with clinicopathological parameters and survival in patients with muscle-invasive transitional cell carcinoma (TCC) of the bladder. METHODS: The data on 108 patients with muscle-invasive bladder TCC, who underwent radical cystectomy, were reviewed retrospectively. Sections were analysed immunohistochemically for D2-40, a specific lymphatic endothelial cell (LEC) marker. Counts of lymph vessels were taken in intratumoural and peritumoural areas as well as in normal tissue. To detect proliferating LECs, we performed a double immunostaining for D2-40 and the proliferation marker Ki-67. RESULTS: Peritumoural vessels were observed in 105 (97.2%) sections and intratumoural vessels in 65 (60.2%). Higher intratumoural lymph vesseI density (LVD) correlated significantly with poor histological differentiation (p=0.01). Higher peritumoural LVD showed a significant association with the presence of lymph node metastasis (p=0.0004). However, LVDs had no statistically significant influence on survival. Intratumoural and peritumoural lymph vessels showed proliferating LECs in varying proportions in all examined samples. CONCLUSIONS: The present study is the first to suggest the existence of proliferating lymph vessels, and, therefore, of lymphangiogenesis in bladder TCC. To our knowledge, it is also the first to confirm a strong correlation of higher peritumoural LVD with the presence of lymph nodes in clinically localized invasive bladder TCC. These findings indicate that lymphangiogenesis may contribute to tumour dissemination and thus provide a potential target for bladder cancer therapy.

Impact of hypoxia and hypercapnia on calcium oxalate toxicity in renal epithelial and interstitial cells.

Although there is an ongoing controversy about the primary site of calcium oxalate stone (CaOx) formation, there is some evidence for extratubular crystallization. However, the mechanisms leading to such interstitial calcifications are not clear. Anatomical studies have demonstrated a close association between the renal vasculature and renal tubules. It has been hypothesized that disorders of the vasculature may contribute to renal stone formation. The exceptional papillary environment with low oxygen and high carbon dioxide is of interest in this context and its impact on CaOx toxicity to renal cells has to be evaluated. LLC-PK1, Madin-Darby canine kidney (MDCK), human umbilical vein endothelial (HUVEC) and fibroblast cell lines were exposed to hypoxia (3% O2) alone, hypercapnia combined with hypoxia (3% O2, 18% CO2) or standard culture conditions (20% O2) for 72 h. Cell survival rates were determined microscopically after 4 h of incubation with CaOx at final concentrations of 1, 2 and 4 mM. DAPI staining and western blot were used to evaluate the induction of apoptosis. We confirmed that CaOx leads to concentration-dependent effects on the viability of the cell lines. HUVECs were most vulnerable to CaOx among the four cell lines. Incubation under hypoxia alone had no impact on CaOx toxicity to any of the cell lines in terms of survival. However, under combined hypoxic and hypercapnic conditions, all cell lines displayed a significant reduction of cell survival compared to room air incubation. Again, this effect was most pronounced for HUVECs. The induction of apoptosis could not be demonstrated in any experimental setting. Combined hypoxia and hypercapnia clearly aggravate CaOx toxicity to renal cell lines. As we could not demonstrate the induction of apoptosis, this effect may be a result of toxic necrosis. Especially the CaOx effect on interstitial cell lines might be of interest in the chronic ischemic papillary environment. An increased toxicity may lead to recurrent stone formation, and vice versa, diseases of the vasculature, like arteriosclerosis, may further promote stone formation by induction of local ischemia. This issue has to be clarified by further studies.