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Alterations of DNA methylation were investigated in 6 urothelial carcinoma cell lines and 13 tumor tissues. The methylation of L1 LINE sequences was diminished in all cell lines (by 26 +/- 5%; range, 11-49%) and in most tumors (by 21 +/- 5%; range, 0-60%) compared to normal bladder mucosa. Hypermethylation of the calcitonin gene CpG island was restricted to cell lines and was not found in primary tumors, suggesting it had arisen during culture. In single-cell clones of a urothelial carcinoma cell line, both hypomethylation of L1 sequences and hypermethylation of the calcitonin gene persisted, indicating that they coexist within one cell. DNA methyltransferase expression did not correlate with the methylation status of the cell lines, but rather with histone H3 expression. Accordingly, it was down-regulated in quiescent cells. Aberrant expression of DNA methyltransferase is therefore not likely the cause for altered methylation patterns in urothelial carcinoma. L1 LINE hypomethylation seems to prevail in urothelial carcinoma and in this tumor might be useful for diagnostic or prognostic purposes.

Objective: Several investigators have demonstrated the high sensitivity of immunocytology in the diagnosis of transitional cell carcinoma (TCC). A new technique, designated "filter immunocytology" (FLIC), simplifies the technique of quantitative immunocytology, considerable decreases assay time and increases the percentage of assessable specimens., Study Design: Voided urine samples were obtained from 89 patients without evidence of TCC and from 91 patients with histologically proven TCC. The cells were transferred onto a polycarbonate membrane. Immunostaining was performed using monoclonal antibody. Due ABC 3, directed against a differentiation antigen on urothelial cells. Specimens containing > 35% positive urothelial cells were regarded as abnormal., Results: Of 153 specimens 180 (85%) were assessable. The investigation of 76 specimens from control patients and 77 from patients with TCC yielded a specificity of 86% and a sensitivity of 75%, respectively. Sensitivity did not correlate with tumor grade. Despite high interobserver and intrapatient variations regarding the amount of antigen-positive cells, a concordant attribution to either "normal" or "abnormal" was made in > 95% of cases. Intraobserver variation was small and did not influence the test result., Conclusion: These results suggest that FLIC assay may be a valuable adjunct to conventional cytology. A careful prospective investigation appears to be worthwhile to further define the indications for this technique.

Recent molecular biology investigations have demonstrated that tumor progression and dissemination in bladder cancer is a highly complicated phenomenon, consisting of multiple distinct steps and regulated by a great number of different genes. Some of these genes involved in the specific steps of tumor progression and dissemination have been identified. Several oncogenes, e.g., the epithelial growth factor receptor (EGF-R), and tumor-suppressor genes, e.g., the p53 gene, have been found to correlate significantly with tumor progression. The decreased expression of cell-adhesion molecules such as E-cadherin appears to facilitate tumor-cell detachment in the primary tumor, whereas expression of the intercellular adhesion molecule (ICAM)-1 might be of relevance for cell attachment at the metastatic site. Tumor invasion through the basement membrane has been correlated with a decreased expression of laminin and elevated urinary levels of acidic fibroblast growth factor. Although the complex processes related to dissemination are far from being completely understood, the finding of differential expression of distinct genes appears to provide the first targets for therapeutic intervention.

Only approximately 20% of the patients with metastatic renal cell carcinoma who undergo systemic immunotherapy will respond to this form of treatment. Appropriate selection of patients could exclude many who will not benefit from immunotherapy from a toxic and expensive treatment. Several factors predicting the outcome of immunotherapy have been reported so far. These predictors can be classified into (1) patient-related factors, (2) tumour-related factors and (3) alterations of the immune system during immunotherapy. Performance status and the interval between tumour nephrectomy and metastasization are the most important patient-related factors. Liver metastases are apparently an unfavourable tumour-related prognostic factor. Various alterations of the immune system during immunotherapy are significantly linked with the outcome of the treatment. In consequence, the prospective investigation of prognostic factors within clinical trials appears to be mandatory.

Recent investigations have demonstrated p53 and Rb alterations in a subset of transitional cell carcinoma (TCC). Further genetic changes during tumor progression include overexpression of the c-myc gene in a significant number of mainly invasive bladder tumors. To study the possible interactions between these genes in TCC, urothelial cancer cell lines were chosen as an in vitro model. Expression and mutation of p53 was studied in 15 bladder cancer cell lines by immunocytochemistry, Western blot, polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing of double stranded PCR products of exons 4, 5, 7 and 8 of genomic DNA. C-myc expression and gene structure were studied using Northern and Southern blot techniques Rb protein expression was analyzed by Western blot. Twelve of 15 cell lines showed either p53 mutations or abnormal protein expression. Consistent with previous studies, five cell lines did not express Rb protein. None of the cell lines studied retained both tumor suppressor genes in a functional form. The c-myc gene appeared to be intact in all cell lines and copy numbers were close to normal. Northern analysis demonstrated that all cell lines expressed c-myc mRNA but evidence for altered regulation was found in at least two cell lines. Our data suggest that amplification or translocation are not the underlying mechanism for c-myc overexpression in urothelial tumors. No correlation between loss of Rb protein and c-myc expression was observed. The results presented here for the cell lines match well those obtained in vivo. Thus, these cell lines may provide a suitable model for further analysis of molecular alterations in urothelial cancer.

The introduction of monoclonal antibodies into the diagnosis of transitional cell carcinoma (TCC) has been a further step toward the use of biological parameters to support conventional cytological diagnosis of this tumor entity. Several investigators have demonstrated the high sensitivity and good specificity of immunocytology. Nevertheless this technique is hampered by the inconvenient and time-consuming microscopic analysis. The purpose of this study was to combine the advantages of immunocytology with the capabilities of an automated flow-cytometric system. Since all monoclonal antibodies (mAbs) currently used for immunocytology cross-react with granulocytes a preselection of urothelial cells becomes necessary for immuno-flow cytometry (immuno-FCM). Based on earlier analyses mAb Due AUT 2, reactive against urothelium and not against granulocytes, was chosen to preselect for urothelial cells. mAb Due ABC 3 was used to discriminate between normal and malignant urothelial cells. Initial experiments including 10 barbotage specimens from patients with histologically proven TCC and concomitant urinary tract infection yielded a high sensitivity (90%) of immuno-FCM. In 10 control patients with malignancies other than bladder TCC and benign diseases ABC 3 expression was not increased. Flow-cytometric examination of irrigation specimens from 10 patients with a history of bladder cancer but without cystoscopic and cytologic evidence of tumor recurrence showed abnormal results in 6 patients. Rebiopsy and/or cystectomy confirmed tumor recurrence in 5 of these patients and in 1 patient with negative immuno-FCM. Ongoing prospective trials will further define the clinical impact of this approach in the diagnosis and follow-up of patients with bladder cancer.

Recent investigations have demonstrated alterations of the p53 tumor-suppressor gene in a considerable number of transitional-cell carcinoma (TCC) specimens. Thus far, these investigations have been restricted to either papillary TCC or invasive bladder cancer. To obtain further information on a possible involvement of p53 in bladder cancer development or tumor progression, investigations of precursor lesions and early stages of this disease are required. Immunohistochemical examination of 6 dysplasias and 24 carcinomas in situ (TIS) showed p53 accumulation, which is suggestive of p53 inactivation, in 2 (33%) and 9 (38%) of these specimens, respectively. This ratio was similar in 9 T1 lesions (33%) and in 14 cases of muscle-infiltrative disease (35%). In papillary tumors, p53 accumulation was observed exclusively in 3/10 moderately differentiated or high-grade lesions but not in 1 Ta G1 tumor. The expression of p53 accumulation was a consistent finding. The examination of tumor recurrences yielded either the presence or the absence of p53 overexpression in the primary and recurrent tumors of 7/8 patients. Similarly, in multifocal TCC, p53 accumulation was also either present or absent in 10/11 cases examined. These results suggest the existence of at least two different subgroups of TCC, with p53 accumulation being present in one of these groups. The observation of p53 accumulation in dysplasia and in TIS is a prerequisite for a possible involvement of p53 in bladder cancer carcinogenesis, although it does not prove this assumption.

Because patients with superficial bladder cancer are in a high-risk group where tumor progression is concerned, topical therapeutic strategies are necessary to prevent tumor recurrence and tumor progression. Based on experimental studies and several case reports, during the last two decades immunotherapy for superficial bladder cancer has been developed. The effects of topical instillation of bacillus Calmette-Guerin (BCG) has been carefully investigated in numerous clinical trials. Especially patients with carcinoma in situ appear to benefit from BCG therapy. Other types of local immunotherapy, e.g., instillation of interferons, interleukins, and keyhole limpet hemocyanin have been found to have fewer side effects than BDG. These new approaches are currently under clinical investigation.

To establish a quantitative dual-parameter flow cytometry (FCM) analysis of cell surface antigens, possible obstacles caused by contaminated leucocytes in a specimen and staining and measuring conditions were investigated using human bladder cancer cell lines, 5637, T24 and SW1710. The first monoclonal antibody (MoAb) used to select urothelial cells in a specimen was applied with the second MoAb used to discriminate between normal and transformed urothelial cells. MoAbs Due AUT 2 and CD45 appeared to be suitable for the selection of urothelial cells, while Due ABC 3 and Due ABC 5 were applied to detect transformed cells. Tumor cell-leucocyte suspension was simultaneously stained with combinations of these MoAbs. The results demonstrated that Due AUT 2 and CD45 effectively eliminated contaminated leucocytes by means of positive and negative selection of the urothelial cells, respectively. Based on these experiments, dual-parameter FCM analyses of bladder washing from 5 patients with bladder cancer were performed using MoAbs Due AUT 2 and Due ABC 3. The results indicated that by dual-parameter FCM distinct antigenic features of transitional cells could be investigated even if considerable amounts of contaminated leucocytes were present. The clinical impact of this approach is a subject of ongoing trials.

The development of the hybridoma technology allows the identification of tumor associated antigens with monoclonal antibodies (mAbs). Employing this technology mAb Due ABC 3 was obtained by immunization of a BALB/c mouse with bladder tumor cell line SW 1710 and subsequent cell fusion of spleen cells with P3. X63.Ag8.653 mouse myeloma cells. MAb Due ABC 3, an IgM antibody, was found to recognize an antigen present in the membrane of tumor cells in 25 out of 28 (89%) transitional cell carcinoma specimens but rarely (three out of 25 specimens, 12%) on normal urothelial cells. Cross reactions were seen with proximal tubular epithelium of the kidney and seven out of 12 renal cell carcinomas examined. Furthermore, the antigen was expressed by granulocytes, some gastrointestinal epithelia, ovarian and breast carcinoma. The antigen recognized by mAb Due ABC 3 was stable to fixation with formaldehyde and paraffin emmbedding, different proteases, alkaline treatment and heat exposure up to 70C. Antigenicity was abandoned by incubation with periodate but not with neuraminidase treatment. The antigen could be extracted with chloroform/methanol suggesting the involvement of a glycolipid. Immuno-thin layer chromatography revealed a single lipid band reacting with mAb Due ABC 3 but not with anti-CD15, directed against the Lewis X antigen. Although not tumor-specific, mAbs directed against differentiation antigens may be of value for the investigation of cell transformations as well as for diagnostic use.

Recently, the development of monoclonal antibody Due ABC 3 directed against transitional cell carcinoma has been reported. With this monoclonal antibody an in vitro test system for diagnosis and followup of patients with transitional cell carcinoma has been developed. The clinical value of this assay, designated as quantitative immunocytology, was evaluated in a prospective trial and compared to conventional cytology. We investigated 74 voided urine specimens obtained from patients with histologically proved transitional cell carcinoma and 60 specimens from donors without clinical evidence of transitional cell carcinoma. Sensitivity was 66% versus 47% for immunocytology and conventional cytology. Specificity of conventional cytology (92%) was higher compared to immunocytology (58%). Statistical analysis demonstrated a significantly higher sensitivity of the combined analysis of conventional cytology and immunocytology (p less than 0.001) compared to conventional cytology alone, without significant differences in specificity. The results obtained with immunocytology were impaired by the great number of nonevaluable specimens. Poor preservation of cells, severe pyuria or an insufficient number of urothelial cells prevented evaluation in 25% of the cases, while only 6% could not be evaluated by conventional cytology. The ability of immunocytology to improve the sensitivity of conventional cytology makes this technique a promising adjunct to the noninvasive diagnosis of transitional cell carcinoma.

In this study, none of the evaluated clinical staging methods was found to predict reliably the presence or absence of extracapsular growth of histologically proved carcinoma of the prostate. In this respect, digital rectal examination, transrectal ultrasound, CT, and MR imaging cannot contribute to treatment decisions in localized prostate cancer. Further studies are under way to determine the value of 7.5-MHz scanners in transrectal ultrasound and high-resolution surface coils in MR imaging.

Functional properties of natural killer cells were analyzed in a 51chromium-release assay by modulating their activity with human beta-interferon using 1) peripheral mononuclear cells of 16 patients with carcinoma of the prostate and 7 healthy male donors, and 2) mononuclear cells from the periprostatic lymph nodes of 6 patients with stage pT2N0M0 prostatic cancer and 5 without malignancy. Cell lines EB 33, PC 3, DU 145 and CaKi 1 were used as target cells. Spontaneous cell-mediated cytotoxicity of peripheral mononuclear cells was depressed in patients with advanced prostatic cancer; however, the natural killer cells responded as those of the healthy controls to beta-interferon. The beta-interferon effect was time and dose dependent. In mononuclear periprostatic lymph node cells virtually no spontaneous cell-mediated cytotoxicity was detected. The reactivity of mononuclear periprostatic lymph node cells from patients with prostatic cancer was significantly less improved by beta-interferon stimulation than the mononuclear periprostatic lymph node cells of healthy subjects. The stimulation of mononuclear periprostatic lymph node cells by beta-interferon was reduced significantly compared to simultaneously tested autologous peripheral mononuclear cells.