Ingo Kausch

A robust methodology to study urine microRNA as tumor marker: microRNA-126 and microRNA-182 are related to urinary bladder cancer.

OBJECTIVES MicroRNAs have been shown to be related to specific types of malignant cell growth. In case of urothelial bladder cancer (BCa), novel noninvasive diagnosis is particularly required and it is attractive to consider, as urine is an easily available source for molecular markers including RNA. In this context, we aimed to develop a clinically applicable and sensitive protocol for the preparation and molecular analysis of low molecular weight RNA from urine samples obtained from bladder cancer patients or healthy volunteers. MATERIALS AND METHODS First, a method was developed for the preparation of low molecular weight RNA from a set of urine samples from different donor groups: (1) patients with low-grade BCa, (2) patients with high-grade BCa, (3) patients with urinary tract infections, (4) healthy donors; each n = 9. The RNA extracts were then used to monitor a number of 157 microRNA species by quantitative reverse transcriptase-polymerase chain reaction. Subsequently, those microRNAs that showed a higher abundance in urine samples from BCa patients were detected in an independent set of urine samples (n = 47). RESULTS The significance and diagnostic usefulness of this methodology is reflected by the finding that the RNA ratio of microRNA-126:microRNA-152 enabled the detection of BCa from urine at a specificity of 82% and a sensitivity of 72%, with an area under the curve of 0.768 (95% confidence interval, 0.605-0.931). CONCLUSIONS This study describes a novel, robust, and useful technology platform that is suitable to analyze small RNAs, including novel RNA-based tumor markers, in urine samples. A detailed technical analysis of this methodology provides new insights into the characteristics of urine microRNA such as composition and the donor-dependent variability.

Photodynamic Diagnosis in Non–Muscle-Invasive Bladder Cancer: A Systematic Review and Cumulative Analysis of Prospective Studies

CONTEXT The clinical benefit of photodynamic diagnosis (PDD) with 5-aminolevulinic acid or hexaminolevulinate in addition to white-light cystoscopy (WLC) in bladder cancer has been discussed controversially. OBJECTIVE To assess in a systematic review the effect of PDD in addition to WLC on (1) the diagnosis and (2) the therapeutic outcome of primary or recurrent non-muscle-invasive bladder cancer investigated by cystoscopy or transurethral resection. EVIDENCE ACQUISITION An electronic database search of Medline, Embase, the Cochrane Library, and CancerLit was undertaken, plus hand searching of relevant congress abstracts and urologic journals. Trials were included if they prospectively compared WLC with PDD in bladder cancer. The review process followed the guidelines of the Cochrane Collaboration. Two reviewers evaluated independently both trial eligibility and methodological quality and data extraction. EVIDENCE SYNTHESIS The primary end point of diagnostic accuracy was additional detection rate. The primary end points of therapeutic outcome were residual tumour at second resection and recurrence-free survival (RFS). Seventeen trials were identified. Twelve diagnostic trials used WLC and PDD with the same patients. Seven reported results for the subgroup of patients with carcinoma in situ (CIS). Five randomised trials studied therapeutic outcome. The results were combined in random effects meta-analyses if end points, designs, and populations were comparable. Twenty percent (95% confidence interval [CI], 8-35) more tumour-positive patients were detected with PDD in all patients with non-muscle-invasive tumours and 39% (CI, 23-57) more when only CIS was analysed. Heterogeneity was present among diagnostic studies even when the subgroup of patients with CIS was investigated. Residual tumour was significantly less often found after PDD (odds ratio: 0.28; 95% CI, 0.15-0.52; p<0.0001). RFS was higher at 12 and 24 mo in the PDD groups than in the WLC-only groups. The combined p value of log-rank tests of RFS was statistically significant (0.00002). CONCLUSIONS PDD detects more bladder tumour-positive patients, especially more with CIS, than WLC. More patients have a complete resection and a longer RFS when diagnosed with PDD.

Molecular aspects of bladder cancer. III. Prognostic markers of bladder cancer

The current pathological and clinical parameters provide important prognostic information, yet still have limited ability to predict the true malignant potential of most bladder tumors. In the last years, investigation of the basic mechanisms involved in carcinogenesis and tumor progression by molecular biology has provided a host of markers which are of potential diagnostic or prognostic value for bladder carcinoma. These markers may serve as tools for early and accurate prediction of tumor recurrence, progression and development of metastases and for prediction of response to therapy. The precise prediction of tumor biological behavior would facilitate treatment selection for patients who may benefit from radical surgical treatment or adjuvant therapy. We provide a current, comprehensive review of the literature on bladder tumor markers with a special emphasis on their prognostic potential. The literature suggests that currently no single marker is able to accurately predict the clinical course of bladder tumors and thus would serve as a reliable prognosticator. A combination of prognostic markers could predict which superficial tumors need an aggressive form of therapy and which invasive tumors require adjuvant therapy. Altogether, the most promising markers are, at this point, Ki-67 and p53 expression as well as matrixmetalloproteinase complex and angiogenesis.

Antisense treatment against Ki-67 mRNA inhibits proliferation and tumor growth in vitro and in vivo.

The Ki‐67 protein is tightly regulated and depends on the proliferative status of a cell. It is present in the nuclei of proliferating cells but absent in resting cells. Since transformation of malignant cells is frequently associated with high cell proliferation and since proliferation is tightly associated with the Ki‐67 protein labeling index, this antigen may represent a potential target for cancer therapy. In the present study we determined the ability of a phosphorothioate antisense oligodeoxyribonucleotide (ODN) targeted against Ki‐67 mRNA to inhibit tumor cell proliferation specifically in cell culture, in multicellular 3‐dimensional spheroids (MCS) and in subcutaneous murine tumor models. Antisense treatment of 1 myeloid and different epithelial tumor cell lines in suspension and monolayer culture, respectively, resulted in specific reduction of Ki‐67 mRNA and protein, inhibition of proliferation and increased apoptotic cell death. Multicellular human bladder carcinoma spheroids lost their 3‐dimensional structure and underwent cell death after incubation with antisense oligonucleotides. The growth of subcutaneous syngeneic prostatic (p = 0.05) and transitional cell tumors (p = 0.001) in immunocompetent mice was significantly inhibited in antisense‐treated animals. From these findings we conclude that antisense inhibition of Ki‐67 protein expression may be a rational approach in anticancer therapy.

The expression of Ki-67, MCM3, and p27 defines distinct subsets of proliferating, resting, and differentiated cells.

The mini‐chromosome maintenance proteins (MCM), which are involved in the control of DNA replication, and the cyclin‐dependent kinase inhibitors, such as p27/KIP1, represent two groups of proteins that are currently under investigation as diagnostic tumour markers. The expression of p27 and MCM3 was compared with the expression of the Ki‐67 protein, an approved marker for proliferating cells, extensively used in histopathology and cancer research. The expression pattern of all three proteins was assessed on germinal centres and oral mucosa, which display a well‐defined spatio‐temporal organization. The expression of the p27 protein was closely related to differentiated cells, whereas MCM3 and Ki‐67 were predominantly localized to the regions of proliferating cells. However, it is important to note that considerable numbers of cells that were growth‐arrested, as confirmed by the absence of the Ki‐67 protein, stained positive for the MCM3 protein. These results were verified in vitro using growth‐arrested Swiss 3T3. The MCM3 protein is therefore expressed in cells that have ceased to proliferate, but are not terminally differentiated, according to the absence of p27 protein expression. In conclusion, a combined analysis of Ki‐67, MCM3, and p27 protein expression may provide a more detailed insight into the cell proliferation and differentiation processes that determine individual tumour growth. Copyright

Prostate Cancer Vaccines

Standard systemic treatment of prostate cancer today is comprised of antihormonal and cytostatic agents. Vaccine therapy of prostate cancer is principally attractive because of the presence of tumor-associated antigens such as prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), prostate-specific membrane antigen (PSMA), and others. Most prostate cancer vaccine trials have demonstrated some activation of the immune system, limited clinical success, and few adverse effects.One strategy to overcome the problem of limited clinical success of vaccine therapies in prostate cancer could be strict patient selection. The clinical course of patients with prostate cancer (even in those with PSA relapse following surgery or radiotherapy with curative intention, or those with metastatic disease) can vary significantly. In patients with organ-confined prostate cancer, the most promising immunotherapeutic approach would be an adjuvant therapy following surgery or radiotherapy. Patients with PSA relapse following surgery or radiotherapy could also benefit from immunotherapy because tumor burden is usually low. However, most patients in prostate cancer vaccine trials had metastatic hormone-refractory prostate cancer (HRPC). High tumor burden correlates with immune escape phenomena. Nevertheless, 2 years ago, it was demonstrated, for the first time, that a tumor vaccine can prolong survival compared with placebo in patients with HRPC. This was demonstrated with the vaccine sipuleucel-T (APC-8015; Provenge®), a mixture of cells obtained from the patient’s peripheral blood by leukapheresis followed by density centrifugation and exposition. The Biologics License Application for this vaccine was denied by the US FDA in mid 2007, however, because the trial had failed to reach the primary endpoint (prolongation of time to tumor progression). Nevertheless, clinical trials with sipuleucel-T are ongoing, and the approach still looks promising. Another interesting approach is a vaccine made from whole tumor cells: GVAX®. This vaccine is presently being studied in phase III trials against, and in combination with, docetaxel. The results from these trials will become available in the near future.Besides the precise definition of the disease status of patients with prostate cancer, combinations of vaccine therapy with radiotherapy, chemotherapy, and/or hormonal therapy are approaches that look promising and deserve further investigation.

Expression of KIT (CD117) in Renal Cell Carcinoma and Renal Oncocytoma

Objective: Overexpression of KIT (CD117), a tyrosine kinase receptor, has been reported in a variety of tumors, some of which are susceptible to therapy with imatinib mesylate. Our aim was to analyze KIT expression immunohistochemically in renal cell carcinomas (RCCs) and in oncocytomas. Methods: Routinely processed, paraffin-embedded specimens from 61 RCCs and 13 renal oncocytomas were investigated immunohistochemically. Cytoplasmic and membrane-bound KIT staining of tumor cells was determined semiquantitatively. A subset of cases was additionally analyzed for point mutations of c-kit exon 17 by peptide nucleic acid-mediated nested polymerase chain reaction-clamping. Results: All cases of oncocytomas and chromophobe RCCs showed membrane-bound KIT positivity, while about three-quarters of cases showed cytoplasmic reactivity. All other types of RCC were found KIT negative. Within the group of chromophobe RCCs, negative cytoplasmatic KIT reactivity was significantly correlated with advanced tumor stage (pT ≧ 2; p = 0.036). Analysis of c-kit exon 17 revealed no ‘gain-of-function’ mutation like the codon 816 Asp→Val mutation (D816V). Conclusions: KIT expression is a hallmark of oncocytoma and chromophobe RCC. Since all other types of RCC were found to be KIT negative, immunohistochemical KIT reactivity may be used as an additional diagnostic criterion to distinguish chromophobe RCC from other RCC types. KIT reactivity and the absence of c-kit mutation D816V in chromophobe RCC justify speculations that imatinib mesylate therapy could be effective in patients with advanced disease.

Antisense oligonucleotide therapy in urology.

PURPOSE Antisense oligonucleotides are short modified DNA or RNA molecules designed to bind selectively messenger RNA and inhibit synthesis of the encoded protein. In the last 20 years antisense technology has emerged as an exciting and promising strategy, especially for treating cancer. We provide urologists with a contemporary review of relevant background information and outline current treatment strategies and clinical trials of antisense oligonucleotide therapy for urological tumors. MATERIALS AND METHODS We comprehensively reviewed the literature, including PubMed and recent abstract proceedings from international meetings, on preclinical and clinical studies of antisense oligonucleotide therapy in urology. RESULTS Current preclinical antisense strategies in urological cancer research include the inhibition of proliferation and induction of tumor cell differentiation, reversal of immunosuppression by tumor secreted molecules and induction of apoptosis. The use of phosphorothioate oligonucleotides as antisense agents has shown promising results in various preclinical cancer models. In recent and current clinical trials in patients with urological tumors antisense agents targeted against c-raf kinase, protein kinase C-alpha, protein kinase A and bcl-2 are being evaluated. CONCLUSIONS Many compounds have achieved convincing in vitro reduction of target messengerRNA and protein expression. Early clinical trials show safety and mild toxicity at the given doses. Overall the current state of antisense oligonucleotide research described promises a highly productive future for this technology. However, for most medical applications of antisense compounds many obstacles related to nuclease stability, affinity, cellular delivery and specificity remain to be clarified.

TREATMENT OF RECURRENT PENILE CONDYLOMATA ACUMINATA WITH EXTERNAL APPLICATION AND INTRAURETHRAL INSTILLATION OF BACILLUS CALMETTE-GUERIN

PURPOSE Condylomata acuminata are caused by human papillomavirus infection. Despite numerous treatment modalities these patients often demonstrate recurrent disease. We report initial experience with bacillus Calmette-Guerin (BCG) therapy in patients not responding to standard treatment. MATERIALS AND METHODS Between October 1994 and March 1997, 6 men with rapidly recurrent external and intraurethral condylomata acuminata underwent BCG therapy after initial laser treatment. External application and intraurethral instillation of BCG were performed 6 times in weekly intervals. Followup studies included examination and endoscopic inspection of the urethra and bladder. RESULTS Of the patients 3 completed 1 course of BCG and had no relapse of condylomata acuminata, 2 underwent a second course of BCG and 1 had relapse, and 1 had relapse after discontinuing therapy due to penile edema. The annual recurrence rate decreased from 3.2 before to 0.75 after BCG therapy (p < 0.05, test of equality of 2 percentages). CONCLUSIONS Immunotherapy with BCG is accepted treatment for superficial transitional cell carcinoma. The BCG induced immune response appears to reduce the recurrence rate in patients with condylomata acuminata.

Primitive Neuroectodermal Tumor (PNET) of the Urinary Bladder

We report on the clinical, morphologic, immunohistochemical, ultrastructural, and molecular cytogenetic features of a primitive neuroectodermal tumor (PNET) primarily arising in the urinary bladder. An 81-year-old man presented with lymphedema of the lower extremities, fatigue, and urge incontinence. Radiographically, a tumor filling the entire cavity of the urinary bladder and extending into the pelvic and retroperitoneal tissue was noted. Histology of tumor biopsies showed a highly cellular, focally necrotic small round-cell tumor with numerous mitoses and occasional rosette-like structures. The tumor cells displayed significant immunoreactivity for neuron-specific enolase (NSE) and the MIC2 gene product (CD99). Dense-core granules were detectable by electron microscopy. A molecular cytogenetic analysis using comparative genomic hybridization (CGH) revealed gains of the chromosomes 3p, 6, 8q, 12, 17q, and 21q. The patient died two weeks after diagnosis. To the best of our knowledge, this is the fifth reported case of a PNET of the urinary bladder, and the first that includes a molecular cytogenetic analysis based on CGH.