B. J. Schmitz-Dräger

P53 accumulation in precursor lesions and early stages of bladder cancer

SummaryRecent 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. Similarily, 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.

Inactivation of tumor suppressor genes and deregulation of the c-myc gene in urothelial cancer cell lines.

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.

Over-expression and amplification of the c-myc gene in human urothelial carcinoma.

To understand the mechanisms underlying increased expression of Myc protein in human urinary bladder cancer, expression of c‐myc mRNA and the copy number of the c‐myc gene were determined. Expression of mRNA was measured by quantitative RT‐PCR in 40 urothelial carcinomas and in 18 histologically normal mucosae. Mean expression in tumors was significantly increased (3.23 ± 2.63 AU vs. 1.90 ± 0.95 AU, p < 0.023) and exceeded the highest level in normal mucosa in 15 (37.5%) tumors. The c‐myc gene copy number was higher than in leukocytes and normal bladder mucosa in 14 of 40 tumors, but only 3 among these showed a more than 4‐fold increase indicative of gene amplification. Most, but not all, tumors with elevated expression displayed an increased gene copy number (p < 0.0001). In line with other studies of the protein level, no significant association either of c‐myc mRNA over‐expression or of increased gene copy number with tumor stage or grade was observed. The data indicate that elevated mRNA expression as a consequence of increases in c‐myc gene copy number often underlies Myc protein over‐expression in bladder cancer. This increase may be a consequence of, most frequently, chromosome 8q gain and, occasionally, gene amplification, while in some tumors deregulation of mRNA expression occurs without evident changes in the c‐myc gene copy number. Int. J. Cancer (Pred. Oncol.) 84:169–173, 1999.

c-myc in bladder cancer Clinical findings and analysis of mechanism

The c-myc gene product is known to be involved in the regulation of cell proliferation and differentiation. Altered c-myc gene expression is a common event in a variety of tumors. This study was designed to investigate c-myc overexpression in transitional cell carcinoma (TCC). The first part was designed to investigate the frequency of c-myc overexpression in relation to tumor stage and tumor grade. A second set of experiments was directed at the mechanisms underlying c-myc overexpression in TCC. A total of 185 paraffin-embedded urothelial tissue specimens were investigated immunohistochemically for c-myc overexpression. A single case of overexpression (6%) was observed in normal urothelial tissue (n=16). c-myc overexpression was also infrequent in carcinoma in situ (TIS) (7/39=18%). In contrast, papillary urothelial tumors (n=65) yielded c-myc overexpression in 38 cases (58%). Investigation of infiltrating bladder tumors revealed c-myc overexpression in 56% of T1 tumors and 59% of muscle-infiltrating tumors. The staining pattern in multifocal tumors was heterogeneous in 10 of 18 cases. Similarly, only 12 of 28 patients with tumor recurrences showed the same c-myc staining pattern in the primary tumorand in tumor recurrences. c-myc overexpression did not correlate with tumor grade or tumor progression. Nevertheless, the high frequency of c-myc overexpression in urothelial carcinoma suggests an important role for this protein in urothelial carcinoma. Therefore, the mechanism underlying c-myc overexpression was further investigated in six bladder carcinoma cell lines. Southern blot experiments under standardized conditions showed no significant gene amplification. The comparison of c-myc mRNA expression to that of histoneH3 as a measure of cell proliferation revealed a moderate correlation (r=0.45) in the six cell lines examined. These data suggest that in accord with its established role as a cell cycle competence factor, c-myc may be necessary but not sufficient for the induction of proliferation in urothelial carcinoma.

Expression of G1-->S transition regulatory molecules in human urothelial cancer.

Growth of cancer cells is characterized by accelerated passage through the cell cycle, which is often caused by deregulation of the G1→S transition. In this study the expression of G1→S transition regulatory molecules was analyzed in 32 transitional cell carcinoma specimens and fifteen normal tissues obtained by cystectomy or nephroureterectomy of mainly locally advanced tumors, as well as six bladder cancer cell lines. Expression of mRNAs for cyclins D1 and D2 and cyclin‐dependent kinases (CDK) 2 and 4 was investigated by quantitative reverse transcription‐poly‐merase chain reaction. Overexpression of cyclin D1 compared to normal mucosa was observed in 3 tumors (9.4%), but in neither of the cell lines. All tumors with overexpression were moderately differentiated (G2) pT1 or pT2 tumors, and thus among the less advanced specimens. Cyclin D2 was not expressed in normal bladder mucosa or in tumors. The expression of CDK4 mRNA varied within the same range in mucosa, tumors, and cell lines. CDK2 mRNA expression varied more strongly and was diminished in individual tumors and in four cell lines. It is concluded that cyclin D1 overexpression can play an important role in the early stage of urothelial tumorigenesis, driving cell proliferation. Ectopic expression of cyclin D2 or amplification of CDK4 does not occur at a significant frequency in urothelial carcinomas. Different expression patterns of cyclin D1 and CDK2 indicate heterogeneity in the mechanisms of G1→S transition deregulation in individual bladder tumors which may elicit differences in their biological and clinical behavior.

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.

Monoclonal Antibody Due ABC 3 Directed against Transitional Cell Carcinoma. II. Prospective Trial on the Diagnostic Value of Immunocytology Using Monoclonal Antibody Due ABC 3

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.

Intravesical treatment of superficial bladder carcinoma with interferons

SummaryThe high incidence of tumor recurrence and the multifocal occurrence of superficial bladder carcinoma have stimulated the idea of intracavitary treatment for therapy and prophylaxis. However, all potent agents have been shown to exhibit severe side effects. Interferons (IFN) are known to inhibit the proliferation of various bladder carcinoma cell lines in vitro. In this study the efficacy of topical administration of IFN was investigated in 10 patients with recurrent superficial bladder carcinoma without evidence of local or distant metastases. The patients had had 1–6 tumor recurrences prior to treatment. Starting 5–8 days following transurethral resection, 5×107 IU IFN alpha-2 (arg) were given intravesically twice a week for 6 weeks. The first examination, including cystoscopy and cytological evaluation of urine, was performed 6 weeks later. Except for occasional urinary tract infections, no side effects were observed. At the first follow-up examination 3 months following the beginning of treatment, tumor recurrence was observed in 6 out of 10 patients. In these patients there was no change regarding tumor stage and grade prior to and following IFN treatment. Since in vitro investigations have demonstrated a correlation between IFN receptor status and growth inhibition, determination of IFN receptors in the membrane of bladder carcinoma might be helpful to those patients who could benefit from IFN treatment.

Monoclonal Antibody Due ABC 3 Directed against Transitional Cell Carcinoma. I. Production, Specificity Analysis, and Preliminary Characterization of the Antigen

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.

Molecular biology of dissemination in bladder cancer — laboratory findings and clinical significance

SummaryRecent 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.