"Cancer stem cells"-lessons from Hercules to fight the Hydra.

Following the initial identification of hematopoietic tumor stem cells, such cells were also found in several solid tumor types. In urology, cancer stem cells have only been found in prostate tumors so far. The concept and detection of tumor stem cells rely heavily on findings derived from stem cell research. Therefore, in addition to identifying and characterizing urologic tumor stem cells, research in uro-oncology should also aim at better understanding the stem-cell biology of urologic organs. Insights in similarities and differences gleaned from these studies could be used to develop strategies for targeted destruction of tumor stem cells while sparing the physiological stem cells. The main target of future curative therapies in uro-oncology must therefore be the central, immortal head of the Hydra, the tumor stem cell.

Genetic variation of Aflatoxin B1 aldehyde reductase genes (AFAR) in human tumour cells.

AFAR genes play a key role in the detoxification of the carcinogen Aflatoxin B(1) (AFB(1)). In the rat, Afar1 induction can prevent AFB(1)-induced liver cancer. It has been proposed that AFAR enzymes can metabolise endogenous diketones and dialdehydes that may be cytotoxic and/or genotoxic. Furthermore, human AFAR1 catalyses the rate limiting step in the synthesis of the neuromodulator gamma-hydroxybutyrate (GHB) and was found elevated in neurodegenerative diseases such as Alzheimer's and dementia with Lewy bodies (DLB). The human AFAR gene family maps to a genomic region in 1p36 of frequent hemizygous deletions in various human cancers. To investigate, if genetic variation of AFAR1 and AFAR2 exists that may alter protein detoxification capabilities and confer susceptibility to cancer, we have analysed a spectrum of human tumours and tumour cell lines for genetic heterogeneity. From 110 DNA samples, we identified nine different amino acid changes; two were in AFAR1 and seven in AFAR2. In AFAR1, we found genetic variation in the proposed substrate-binding amino acid 113, encoding Ala(113) or Thr(113). An AFAR2 variant had a Glu(55) substituted by Lys(55) at a position that is conserved among many aldo-keto reductases. This polarity change may have an effect on the proposed substrate binding amino acids nearby (Met(47), Tyr(48), Asp(50)). Further population analyses and functional studies of the nine variants detected may show if these variants are disease-related.

Malignant advanced granulosa cell tumor of the adult testis: case report and review of the literature.

Testicular granulosa cell tumors (GCTs) are very rare neoplasms. Although adult GCTs are thought to have a relatively indolent course, several reports have demonstrated the malignant potential of these lesions. In case of distant metastases, the overall survival is very short. To date, there is no well-established treatment for these tumors owing to poor results and very rapid progression. A 55-year-old male patient was diagnosed with a testicular GCT with distant lung metastases. He underwent surgical treatment with orchiectomy and adjuvant polychemotherapy (cisplantine, etoposide, and bleomycine) as well as metastasectomy of the right lung. We report the first case of a successfully treated testicular GCT with bipulmonary metastases at initial diagnosis. Thirty-nine months after treatment, the patient is alive with no evidence of disease. We subsequently reviewed all reported cases of an adult GCT in the published literature (25 published cases). This review will summarize all reported cases and discuss treatment options. The current case suggests that a combination of varying treatment modalities could be a promising and reasonable way to manage malignant advanced GCT of the adult testis.

Clinical and functional results after continent cutaneous urinary diversion with the ileal double-T-pouch.

INTRODUCTION: The aim of the study was to assess the clinical and functional results after continent cutaneous diversion with the ileal double-T-pouch. PATIENTS AND METHODS: Between July 1998 and July 2006, 19 patients underwent continent urinary diversion with a cutaneous ileal double-T-pouch. Follow-up investigations included blood chemistry, renal ultrasound and evaluation of the outlet function. The median follow-up was 24 months (range 3-76). RESULTS: There were no intraoperative complications and no perioperative mortality. Early postoperative complications not related to urinary diversion occurred in 6 patients (31.6%). Five patients developed complications related to the urinary diversion (26.3%). The mean operating time was 580 min (420-840). Including the patients who had good results after reoperation, 16 out of the 19 patients (84.2%) were continent day and night without any catheterization difficulties. The average pouch capacity was 490 ml (range 250-1,100). A mild acidosis was the only metabolic disorder observed. During follow-up renal function remained stable in all patients, urinary reflux, recurrent pyelonephritis or stone formation were not found. Three patients with a body mass index >30 developed a necrosis of the efferent loop resulting in pouch cutaneous fistulas. CONCLUSIONS: Continent cutaneous urinary diversion with the ileal double-T-pouch led to good intermediate functional and clinical results. However, construction of the pouch is sophisticated, resulting in a long operation time. The double-T-pouch is not recommended in obese patients.

A new and reliable culture system for superficial low-grade urothelial carcinoma of the bladder.

Several bladder cancer culture systems have been developed in recent years. However, reports about successful primary cultures of superficial urothelial carcinomas (UC) are sparse. Based on the specific growth requirements of UC described previously, we developed a new and reliable culture system for superficial low-grade UC. Between November 2002 and April 2006, 64 primary cultures of bladder cancer specimens were performed. After incubating the specimens overnight in 0.1% ethylenediaminetetraacetic acid solution, tumour cells could easily be separated from the submucosal tissue. Subsequently, cells were seeded in a low-calcium culture medium supplemented with 1% serum, growth factors, non-essential amino acids and glycine. The malignant origin of the cultured cells was demonstrated by spectral karyotyping. Overall culture success rate leading to a homogenous tumour cell population without fibroblast contamination was 63%. Culture success could be remarkably enhanced by the addition of glycine to the culture medium. Interestingly, 86.4% of pTa tumours were cultured successfully compared to only 50% of the pT1 and 38% of advanced stage tumours, respectively. G1 and G2 tumours grew significantly better than G3 tumours (86, 73 and 41%, respectively). Up to three passages of low-grade UC primary cultures were possible. We describe a new and reliable culture system, which is highly successful for primary culture and passage of low-grade UC of the bladder. Therefore, this culture system can widely be used for functional experiments on early stage bladder cancer.

Heparin-binding epidermal growth factor-like growth factor isoforms and epidermal growth factor receptor/ErbB1 expression in bladder cancer and their relation to clinical outcome.

BACKGROUND: Cleavage of membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields a soluble HB-EGF isoform (sHB-EGF), which is an activating epidermal growth factor receptor (EGFR) ligand and a C-terminal fragment HB-EGF-C acting directly in the nucleus. In bladder cancer, overexpression of both HB-EGF and EGFR have been observed, but to the authors' knowledge the prognostic significance of different modes of HB-EGF signaling have remained unclear. METHODS: Expression and intracellular localization of HB-EGF and EGFR were examined by immunohistochemistry in paraffin-embedded specimens from 121 patients who underwent cystectomy for bladder cancer. Tumor stage was pTis/pT1 in 7 patients, pT2 in 41 patients, pT3 in 55 patients, and pT4 in 18 patients. Lymph node metastases were present in 32 patients. RESULTS: Using an antibody directed against the C-terminal domain, HB-EGF expression was detected in the cytoplasm or in the nucleus of tumor cells. EGFR staining was uniform at the plasma membrane. The actuarial 5-year cancer-specific survival of patients with tumors with predominant nuclear HB-EGF staining was 28% compared with 57% if HB-EGF staining was predominantly cytoplasmic (P = .027). Disease outcome of patients with a 'mixed' HB-EGF staining pattern was found to be between that of the 2 former groups. In agreement with previous studies, strong EGFR expression was associated with poor prognosis. Despite strong EGFR expression, predominant cytoplasmic HB-EGF staining was associated with a more favorable outcome, whereas a predominant nuclear pattern defined a subgroup with extremely poor prognosis (5-year tumor-specific survival of 55% vs 13%, respectively; P = .026). CONCLUSIONS: The current study results confirm that EGFR expression is significantly correlated with disease-specific mortality but that the outcome is also influenced by the mode of HB-EGF signaling. Additional nuclear HB-EGF signaling, indicative of increased cleavage of proHB-EGF, appears to enhance the adverse activities. Cytoplasmic HB-EGF staining likely reflects proHB-EGF, which may also exert antiproliferative effects.

In situ detection of global DNA hypomethylation in exfoliative urine cytology of patients with suspected bladder cancer.

Global DNA hypomethylation is a common phenomenon in bladder cancer. Therefore we investigated whether it is possible to detect and assess global DNA hypomethylation in bladder cancer using a specific monoclonal antibody for 5-methyl-cytosine. Cytospins from exfoliative urine cytology specimens of patients with bladder cancer or a history of bladder cancer, control patients with benign urological diseases and of young healthy volunteers were analyzed. Urothelial carcinoma (UC) cells showed various degrees of nuclear destaining indicating global DNA hypomethylation whereas all specimens from healthy volunteers showed granular nuclear staining indicating regular methylation of repeated DNA sequences. Lowest 5-methylcytosine immunostaining scores were observed in carcinoma cells and a statistically significant difference was observed between urothelial cells of healthy controls or patients with benign disease compared to bladder cancer patients (p<0.01, p<0.05, respectively). In UC cases even morphologically normal urothelial cells often displayed evident hypomethylation. Likewise, in patients with a history of UC, but no cystoscopic evidence of recurrence, morphologically non-malignant urothelial cells presented with some degree of demethylation. Our results strongly support the hypothesis of early global demethylation in bladder cancer. Immunocytochemical staining with the 5-methylcytosine antibody allows simultaneous individual assessment of nuclear morphology and methylation status of a given sample.

Penile metastasis secondary to follicular thyroid carcinoma.

Penile metastases are rare and are considered to reflect end-stage malignant disease. The first case of a follicular thyroid carcinoma metastasizing to the penis is described. Local tumor control and probably enhanced survival was achieved by extended surgery of a previous pelvic recurrence and the penile metastasis and this procedure may be justified in selected cases.

Identifying superficial, muscle-invasive, and metastasizing transitional cell carcinoma of the bladder: use of cDNA array analysis of gene expression profiles.

PURPOSE: Expression profiling by DNA microarray technology permits the identification of genes underlying clinical heterogeneity of bladder cancer and which might contribute to disease progression, thereby improving assessment of treatment and prediction of patient outcome. EXPERIMENTAL DESIGN: Invasive (20) and superficial (22) human bladder tumors from 34 patients with known outcome regarding disease recurrence and progression were analyzed by filter-based cDNA arrays (Atlas Human Cancer 1.2; BD Biosciences Clontech) containing 1185 genes. For 9 genes, array data were confirmed using real-time reverse transcription-PCR. Additionally, Atlas array data were validated using Affymetrix GeneChip oligonucleotide arrays with 22,283 human gene fragments and expressed sequence tags sequences in a subset of three superficial and six invasive bladder tumors. RESULTS: A two-way clustering algorithm using different subsets of gene expression data, including a subset of 41 genes validated by the oligonucleotide array (Affymetrix), classified tumor samples according to clinical outcome as superficial, invasive, or metastasizing. Furthermore, (a) a clonal origin of superficial tumors, (b) highly similar gene expression patterns in different areas of invasive tumors, and (c) an invasive-like pattern was observed in bladder mucosas derived from patients with locally advanced disease. Several gene clusters that characterized invasive or superficial tumors were identified. In superficial bladder tumors, increased mRNA levels of genes encoding transcription factors, molecules involved in protein synthesis and metabolism, and some proteins involved into cell cycle progression and differentiation were observed, whereas transcripts for immune, extracellular matrix, adhesion, peritumoral stroma and muscle tissue components, proliferation, and cell cycle controllers were up-regulated in invasive tumors. CONCLUSIONS: Gene expression profiling of human bladder cancers provides insight into the biology of bladder cancer progression and identifies patients with distinct clinical phenotypes.

Inhibition of p53 function diminishes androgen receptor-mediated signaling in prostate cancer cell lines.

Current therapy for advanced prostate cancer is mainly based on androgen deprivation, although most patients relapse to androgen-insensitive disease. Several mechanisms contributing to androgen-independent growth including alterations in the structure or expression of the androgen receptor (AR) and its cofactors have been identified. Recent evidence suggests that p53 is involved in androgen signaling. The analysis of the effect of p53 on androgen signaling was performed in 22Rv1 and LNCaP prostate cancer cells that express both p53 and AR. The overexpression of p53 diminished the androgenic response in both cell lines in a reporter gene assay. Conversely, the inhibition of p53 by three different p53 inhibitors, Pifithrin-1alpha (PFT-1alpha), an inhibitor of p53-dependent transactivation; MDM2, a regulator of p53 expression; and a dominant-negative N-terminally truncated p53 gene also reduced transactivation of androgen-dependent reporter genes. The inactivation of p53 by PFT-1alpha decreased AR-protein expression in both 22Rv1 and LNCaP cells. Our findings confirm that the overexpression of wild-type p53 decreases androgen function, whereas p53 expression at physiological levels stabilizes AR signaling. Thus, our findings suggest that there is a balance of AR and p53 expression during the androgen-dependent growth of prostate cancer, which is obliterated during further progression of the disease.

Detection of circulating prostate cells during radical prostatectomy by standardized PSMA RT-PCR: association with positive lymph nodes and high malignant grade.

BACKGROUND: The purpose of this study was to establish a quantitative standardized RT-PCR for the detection of Prostate Specific Membrane Antigen (PSMA)-expressing circulating cells and to evaluate clinical relevance in patients undergoing radical retropubic prostatectomy (RRP) for clinically localized prostate cancer (PCa). MATERIALS AND METHODS: An external standard molecule (PSMA MIMIC) was constructed for standardization of PSMA RT-PCR reactions. It has the same sequence as endogenous PSMA, except for a central 85 bp deletion, allowing the amplification of both targets simultaneously with nearly the same amplification characteristics as in a nested PCR. PSMA RT-PCR was performed from peripheral blood samples of 73 patients with clinically localized PCa, 4 with metastatic PCa, 27 with benign prostatic hyperplasia (BPH) and 27 controls. Pre, intra- and postoperative blood samples were tested for circulating PSMA expressing cells in 54 out of 73 patients with clinically localized PCa. We also tested intraoperative blood samples of 19 BPH patients treated by transurethral or open surgery. RESULTS: Endogenous PSMA signals from PCa patients varied between 850 and 9900 transcript molecules, corresponding to 2-20 PSMA-expressing cells/ml blood. Standardized RT-PCR using the PSMA MIMIC molecule revealed a significant decrease of "false-positives" in cancer-free controls (p = 0.004). Controls could clearly be distinguished from prostate cancer patients based on PSMA PCR positivity (p = 0.003). Thirty-two % of patients with localized prostate cancer, 11% of BPH patients and 7% of healthy controls were positive in standardized assays compared to 48%, 30% and 27% without PSMA MIMIC, respectively. Preoperatively, a correlation with tumor stage (p = 0.030), grade (p = 0.035) and Gleason Score (p = 0.03) could be demonstrated in clinically localized PCa patients. Dissemination of prostate cells during surgery occurred in 32% of the RRPs and 21% of BPH patients. Positive PCR signals from intraoperative blood samples correlated with positive lymph node status (p = 0.007) and tumor grade (p = 0.005). Postoperative positive results correlated with grade (p = 0.012) and Gleason Score (p = 0.035). CONCLUSION: Counseling the patient with clinically localized prostate cancer can be challenging. Surgery may be, in retrospect, inappropriate in a number of patients due to preoperative understaging. This newly constructed external standard allows quantitative detection of circulating prostate cells, and therefore may open new perspectives for PSMA RT-PCR techniques as diagnostic assays and tools for post-therapeutic follow-up.

Effect of routine repeat transurethral resection for superficial bladder cancer: a long-term observational study.

PURPOSE: We determined the long-term outcome in patients with superficial bladder cancer (Ta and T1) undergoing routine second transurethral bladder tumor resection (ReTURB) in regard to recurrence and progression. MATERIALS AND METHODS: We performed an inception cohort study of 124 consecutive patients with superficial bladder cancer undergoing transurethral resection and routine ReTURB (83) between November 1993 and October 1995 at a German university hospital. Immediately after transurethral resection all lesions were documented on a designed bladder map. ReTURB of the scar from initial resection and other suspicious lesions was performed at a mean of 7 weeks. Patients were followed until recurrence or death, or a minimum of 5 years. RESULTS: Residual tumor was found in 33% of all ReTURB cases, including 27% of Ta and 53% of T1 disease, and in 81% at the initial resection site. Five of the 83 patients underwent radical cystectomy due to ReTURB findings. The estimated risk of recurrence after years 1 to 3 was 18%, 29% and 32%, respectively. After 5 years 63% of the patients undergoing ReTURB were still disease-free (mean recurrence-free survival 62 months, median 87). Progression to muscle invasive disease was observed in only 2 patients (3%) after a mean observation of 61 months. CONCLUSIONS: These data suggest a favorable outcome regarding recurrence and progression in patients with superficial bladder cancer who undergo ReTURB. ReTURB is suggested at least in those at high risk when bladder preservation is intended.

Clinical-scale generation of dendritic cells in a closed system.

Immunotherapy of malignant diseases based on dendritic cells (DCs) pulsed with tumor antigens is a promising approach. Therefore, there is a demand for large-scale, clinical-grade ex vivo generation of DCs. Here, a procedure is presented that combines monocyte selection and tissue culture in closed systems under current good manufacturing practice conditions. Leukocytes from three patients with urologic cancers were collected by leukapheresis and subjected to immunomagnetic enrichment. From leukapheresis products containing 1.6 +/- 0.2 x 1010 (mean +/- SEM) leukocytes with a frequency of CD14+ monocytes of 18.7 +/- 2.3%, monocytes were enriched to 94.3 +/- 2.2%. CD14+ cell recovery was 67.0 +/- 4.7%. After 6 days of culture in Teflon bags in X-Vivo 15 medium supplemented with autologous plasma, GM-CSF, and IL-4, cells showed an immature DC phenotype and efficient antigen uptake. Following an additional 3 days of culture in the presence of GM-CSF, IL-4, IL-1beta, IL-6, TNFalpha, and PGE(2), cells (82.0 +/- 5.8% CD83+) displayed a mature DC morphology and phenotype, including expression of CD11b, CD11c, CD18, CD25, CD40, CD54, CD58, CD80, CD86, HLA class I, and HLA-DR as well as expression of CCR7 but not CCR5. The mature DC phenotype remained stable for at least 5 days in the absence of cytokines. Yield of DC was 14.0 +/- 4.7% and viability was 91.9 +/- 3.5%. Mature DCs effectively clustered with naive T cells and potently induced allogeneic T-cell proliferation and IL-2 and IFNgamma but not IL-4 production. Thus, this procedure allows large-scale generation of stably mature, Th1 responses inducing DCs under cGMP conditions in a closed system from cancer patients and is therefore well suited for immunotherapy.

p21 and p53 Immunostaining and survival following systemic chemotherapy for urothelial cancer.

INTRODUCTION: Induction of apoptosis and regulation of cell cycle checkpoints are important mechanisms of chemotherapy-induced cell death. The intact p53 tumor suppressor gene is required for efficient activation of apoptosis. The WAF1/p21 gene is transcriptionally activated by p53 and mediates p53-dependent G1 arrest following DNA damage. Therefore, p53 and p21 expression might be related to urothelial tumor response to cytotoxic therapy. METHODS: In a retrospective study, archival tumor specimens from 60 patients treated with cisplatinum-based systemic chemotherapy for locally advanced and/or metastatic urothelial cancer were immunohistochemically stained for p53 and p21. Response to chemotherapy and overall survival were correlated with the results of immunohistochemistry. RESULTS: Thirty-five tumors (58%) of the 60 specimens showed p53 accumulation, and 25 (42%) expressed detectable p21. No association between p53 accumulation and expression of p21 was observed. Correlation with complete and partial remissions following inductive chemotherapy (n = 39) demonstrated that patients with intact p53 responded significantly better (70 vs. 31%, p < 0.05). However, no difference in overall survival was observed with regard to p53 immunostaining (median 12 and 17 months for p53-positive and p53-negative tumors, respectively). The p21 expression was related neither to response nor to overall survival following inductive chemotherapy. In patients receiving adjuvant chemotherapy after cystectomy (n = 21), the outcome was correlated with the immunohistochemistry results. While the survival times for p53-negative patients (60 months) and p53-positive patients (23 months) did not translate into a significant difference, the median overall survival for patients with p21-positive or p21-negative tumors (60 vs. 21 months) was significantly different (p < 0.005). CONCLUSIONS: The short survival of patients with metastatic bladder cancer may conceal putative differences between different prognostic groups in smaller trials. In contrast, p21 immunohistochemistry appears to be of prognostic value in patients receiving systemic adjuvant chemotherapy for locally advanced bladder cancer. The observations made in this retrospective study in a limited number of patients warrant further investigation on the correlation between G1/S checkpoint regulatory genes and adjuvant chemotherapy in larger prospective studies.

Refined mapping of allele loss at chromosome 10q23-26 in prostate cancer.

BACKGROUND: Allele loss of at least two segments in 10q, one mapping to the PTEN gene and one more distal were described in prostate cancer, with loss more frequent in advanced prostate cancer. METHODS: A 63 cM region from 10q23 to q26 was studied for allele loss (LOH) in 59 prostate cancer samples using a dense map of microsatellite markers. RESULTS: LOH of at least one marker in 10q was observed in 13/59 tumors. LOH increased with grade and stage. Detailed deletion mapping identified three regions of allele loss. The first region mapped to the site of the PTEN gene, the second is defined by loss of one marker, D10S1692, in one tumor, and the third is defined between markers D10S1757 and D10S587, including DMBT, with a subregion of approximately 1.2 Mb mapping between markers D10S209 and D10S1679, lost in one tumor. CONCLUSIONS: LOH at the PTEN gene is frequent but mutations in the remaining allele were not detected by SSCP-screening. There may be more than two tumor suppressor (TS) genes mapping more distal of PTEN. The site for these putative TS genes can now be mapped with a dense set of precisely localized markers in a larger series of advanced tumors.