Flow cytometric DNA analysis after immunoselection of bladder tumor cells with monoclonal antibody DU83.21.

Adequate preservation of neoplastic cells and the elimination of interference by inflammatory cells in measuring tumor cell DNA content represent two important objectives necessary for accurate flow cytometric analysis of bladder carcinomas. An experimental model consisting of a mixture of cultured bladder carcinoma cells (T24) and human buffy-coat (BC) cells was used to evaluate various preservatives and an anti-bladder carcinoma monoclonal antibody (MoAb), DU83.21, for separating inflammatory cells (BC cells) from T24 cells. A final concentration of 25% ethanol was found to be the most effective preservative of several tested. After incubation with the MoAb DU83.21 and propidium iodide (DNA stain), the T24 cells could be separated from the BC cells, permitting accurate DNA analysis of the tumor cells. Application of this system to specimens from bladder cancer patients enhanced the detection and DNA analysis of the tumor cell populations.

Rhodamine 123 phototoxicity in laser-irradiated MGH-U1 human carcinoma cells studied in vitro by electron microscopy and confocal laser scanning microscopy.

Rhodamine 123 (R123) is a permeant, cationic, fluorescent dye that localizes preferentially within mitochondria of living carcinoma cells. MGH-U1 human bladder carcinoma cells incubated in vitro with 10 microM R123 for 30 min and then irradiated at 514.5 nm with an argon ion laser underwent selective, phototoxic injury to mitochondria. Ultrastructurally, treatment with R123 plus irradiation with 10 J/cm2 caused selective, progressive mitochondrial alterations consisting of disruption of cristae, vacuolization, swelling, increasing numbers of ring-shaped and angulated mitochondria at 4 to 8 h after irradiation, and obliteration of many mitochondria at 24 to 48 h. Confocal laser scanning microscopy after treatment with R123 plus irradiation with 10 to 30 J/cm2 demonstrated altered uptake and localization of subsequently administered R123, accompanied by striking mitochondrial fragmentation. Irradiation caused a dose-dependent depletion of extractable R123, due to a photosensitized efflux that began immediately and progressed by 4 h after irradiation with 10 to 30 J/cm2; further uptake after reincubation in the presence of R123 was also quantitatively impaired in cells previously irradiated with 30 J/cm2.

Flow cytometric analysis of primary and metastatic bladder cancer.

A total of 22 patients with high grade P2-4N+ transitional cell carcinoma of the bladder underwent flow cytometric analysis of nuclei obtained from paraffin embedded specimens from the primary (bladder) and metastatic (lymph node) sites. Tumor heterogeneity was defined as polyclonal aneuploidy of the primary tumor (not identified in the population studied) or as a difference in the deoxyribonucleic acid index of the primary and metastatic sites of 0.20 or more (8 patients). With these criteria 8 patients (36%) had heterogeneous tumors and 14 (64%) had homogeneous tumors. The median survival of 14 patients with aneuploid and 8 with diploid primary tumors was 17.5 and 8.0 months, respectively (p equals 0.08, Lee-Desu test). When patient survival was compared to the ploidy of the metastatic site, or in patients with diploid primary and metastatic lesions versus deoxyribonucleic acid aneuploidy at either the primary and/or metastatic site, the aneuploid tumors had a longer survival but this difference was not significant (p equals 0.13 and 0.23, respectively). Our study demonstrates the value of flow cytometry to identify primary metastatic tumor heterogeneity. It also suggests that the presence of metastasis may be a more important factor to define the biological potential of transitional cell carcinoma than is deoxyribonucleic acid ploidy.

[Blood and urine determinations of tissue polypeptide antigen in patients with bladder carcinoma]. / Determinazione sierica ed urinaria del Tissue Polypeptide Antigen (TPA) nei pazienti affetti da carcinoma vescicale.

The Tissue Polypeptide Antigen (TPA) is an oncofetal antigen widely used in the diagnosis and follow-up of several urothelial cancers. Its urinary and serum detection is performed by means of RIA technique. We determined urinary and serum TPA in 30 patients with bladder cancer who underwent a transurethral resection. Ten out of 30 patients were correctly diagnosed by serum TPA, 22 by urinary TPA. The ANOVA test showed a statistically significant correlation between grade and urinary TPA between stage and serum TPA. Urinary TPA showed a good sensibility in low grade and moreover in Ta stage carcinoma. Serum TPA increased its performance with higher grade carcinoma and in presence of a muscle infiltration, but it never reached a sufficient sensibility to be considered a bladder cancer marker. In conclusion the simultaneous determination of urinary and serum TPA does not give more information than the urinary determination alone.

Computerized morphonuclear cell image analyses of malignant disease in bladder tissues.

We analyzed the relationship between several morphonuclear parameters related to nuclear size, densitometry (deoxyribonucleic acid content and ploidy) and the chromatin pattern versus the histopathological grading of 46 bladder cancer samples graded according to the World Health Organization classification. We used a SAMBA 200 cell image processor with software allowing for the discrimination of 15 different parameters on Feulgen-stained imprint smears. In addition, we set up preliminary data banks that enable objective and reproducible grading of unknown cases. This approach must be validated in a large series of cases to create an expert system for bladder malignancy diagnosis.

Human chorionic gonadotropin, neuron specific enolase and deoxyribonucleic acid flow cytometry in patients with high grade bladder carcinoma.

Biopsies from 64 patients with transitional cell carcinoma of the bladder (World Health Organization grade 3 and undifferentiated) were studied with deoxyribonucleic acid flow cytometry of fresh tissue and immunohistochemical staining on the histopathological slides for the presence of neuron specific enolase and human chorionic gonadotropin. No correlation was found among the presence of neuron specific enolase or human chorionic gonadotropin and T category, deoxyribonucleic acid ploidy, percentage of cells in the S phase, presence of metastatic disease or response to therapy. The prognosis for patients with muscle invasive disease and tumors positive for neuron specific enolase or human chorionic gonadotropin was similar to that for patients with tumors negative for these substances. When a possible new marker or prognostic factor is evaluated, it is important to investigate whether the new marker adds information on prognosis to what already is known by established standard methods. Further studies are needed to evaluate the clinical importance of human chorionic gonadotropin (and neuron specific enolase) as a marker in urothelial cancer with regard to prognosis and response to therapy.

Transferrin receptor expression in primary superficial human bladder tumours identifies patients who develop recurrences.

A group of 65 patients with superficial bladder carcinoma was followed for 2 years and tumour recurrence rate was correlated both with transferrin receptor status of the initial primary tumour and with the results of voided urine cytology. Nine of 24 patients with transferrin receptor negative tumours had recurrences compared with 30 of 41 patients with transferrin receptor positive tumours. This difference was highly significant. Urine cytology at presentation was also predictive of further tumour formation: of 30 patients who were transferrin receptor positive and had positive urine cytology, 25 developed recurrences.

The epidermal growth factor receptor and the prognosis of bladder cancer.

Epidermal growth factor is found in high concentrations in urine, and its receptor (EGFr) has been identified in certain bladder tumors. This study was performed to determine whether receptor positivity in the tumor was associated with a poor clinical outcome. One hundred one patients with newly diagnosed bladder cancer were studied prospectively by immunohistochemical staining for the EGFr. There were 76 men and 25 women, with a mean follow-up of 30 months; 49 had tumors invading muscle: 18 were pTl (tumor invading lamina propia) and 34 were pTa (tumor confined to urothelium). Strong staining for the EGFr was found in 48% of tumors and was associated with high stage (P less than 0.001). Death of bladder cancer (40 of 101) was associated independently with high stage (P less than 0.0001) and EGFr positivity (P less than 0.001). In patients with pTa and pTl tumors, EGFr positivity was associated with multiplicity (P less than 0.01), time to recurrence (P less than 0.03), and recurrence rate (P less than 0.004). Tumor progression was associated with EGFr positivity (P less than 0.0001) and multiplicity (P less than 0.05). EGFr were found on a significant proportion of bladder tumors: such tumors were more likely to result in death, recurrence, and progression.

Fibronectin distribution in normal and malignant urothelium.

Fibronectin is a glycoprotein that mediates the attachment of BCG to the murine bladder. To assess the potential role of fibronectin on bladder cancer cells as a specific substrate for BCG binding in man, a semi-quantitative method was employed to evaluate the presence of fibronectin on normal urothelium and bladder cancer. Monoclonal anti-fibronectin binding to normal and malignant urothelial tissues was evaluated by an immunoperoxidase assay. Human tumor cell lines were evaluated with mixed hemadsorption and immunoperoxidase assays. In both systems, immunoreactive fibronectin had low expression on unfixed normal and malignant urothelium. With fixation, immunoreactive fibronectin decreased on supporting stroma and increased in normal and malignant urothelium. Fibronectin distribution did not show tumor specificity either with fixed or unfixed specimens.

Three quantitative assays for human erythroid burst-promoting activity of recombinant growth factors and of omentum-conditioned medium.

Human erythroid burst-promoting activity (BPA) of recombinant growth factors and crude materials, of media conditioned by omentum tissue (OMCM), and of media conditioned by the bladder carcinoma cell line (HTB9CM) was measured by three different culture methods. Using the two-stage culture method, significant activity was shown in OMCM (137%-329% of the control), HTB9CM (102%-333%), recombinant human (rh) granulocyte-macrophage colony-stimulating factor (rhGM-CSF) (179%-220%), rh interleukin 3 (rhIL-3) (232%-676%), and rh insulin-like growth factor 1 (rh IGF-1) (106%-175%), whereas there was no significant increase in the number of erythroid bursts by the same additives when the one-stage culture or the delayed erythropoietin method was employed. Linear dose-response curves were observed in the tested range of rhIL-3 and rhGM-CSF. We also observed that 1) a larger amount of rhGM-CSF was required for the optimal stimulation of erythroid burst-forming units (BFU-E) than for the optimal stimulation of granulocyte-macrophage colony-forming units (CFU-GM), and 2) even the maximum dose of rhGM-CSF increased erythroid bursts to a lesser extent than was possible by the addition of rhIL-3. The former results implies that BPA is not the major activity of GM-CSF, and the latter result, although it is not conclusive, suggests that the GM-CSF-responsive BFU-E represent only a subset population of BFU-E responsive to IL-3. The two-stage culture is a useful assay method for screening BPA in biological materials with respect to accuracy, dose responsiveness, and reproducibility.

Clinical cancer progression in urinary bladder tumors evaluated by multiparameter flow cytometry with monoclonal antibodies. Laval University Urology Group.

Multiparameter flow cytometry studies were performed on clinical samples of human bladder tumors to simultaneously analyse DNA content and the expression of surface glycoproteins defined by monoclonal antibodies T16, Om5, T43, and T138. The results of tests performed on 80 samples of bladder irrigations and tumors from 68 patients were correlated with clinical findings at the time of sampling and with disease outcome prospectively (mean follow-up 2 years). Measuring the level of the panurothelial antigen T16 provided more precision in DNA analysis and served as an internal standard to measure the relative expression of the other cell surface antigens studied. The panel of monoclonal antibodies improved the analytical capacity to study the heterogeneity of antigenic phenotypes within individual samples. Aneuploidy frequently correlated with high stage cancers and with a high rate of clinical cancer progression defined as metastasis or death by cancer. However ploidy was not an entirely reliable prognostic indicator since a significant proportion of Ta and T1 nonprogressing tumors were aneuploid, while in 6/20 cases of cancer progression, the samples were near diploid. Contrary to Om5, T43 and T138 antigens were expressed significantly more often on aneuploid samples, although they appear to provide additional information. T138 was positive on 17/18 samples from patients with high stage cancers of which five were near diploid. It was also positive on 4/5 samples from patients with Ta and T1 tumors in whom disease progressed to metastasis and death. Overall, the expression of T138 antigen was a better single indicator of clinical cancer progression than was ploidy. Further stratification was obtained with combined results of DNA and T138 antigen studies. Within the near diploid group, the incidence of bladder cancer death was 0/26 for T138 negative and 5/13 (38%) for T138-positive patients (P less than 0.01). In the aneuploid group incidence of bladder cancer death was 2/10 (20%) for T138-negative and 12/19 (63%) for T138-positive patients (P less than 0.05). These results suggest that simultaneous flow cytometry measurements of DNA and surface antigens may better assess the prognostic behavior of human bladder tumors.

Correlation of cytokeratin patterns with histopathology during neoplastic progression in the rat urinary bladder.

The discrimination of atypical (premalignant) cells from invasive neoplastic cells in primary bladder lesions is a major diagnostic problem in cytopathology and surgical pathology. We have used an animal model of urinary bladder carcinogenesis to determine the specific changes which occur in the expression of certain cytokeratins (CK) during the progression of lesions from regenerative hyperplasia and carcinoma in situ to transitional cell carcinomas. At sequential time points following exposure of the rat bladder epithelium to N-methyl-N-nitrosourea in vivo, immunohistochemical staining of CKs was evaluated in ethanol-fixed samples from the induced urothelial lesions using commercially available anti-CK mouse monoclonal antibodies. Specific changes were found in the expression of CKs 13, 18, and 19 during the neoplastic progression of induced urothelial lesions in the rat. These changes included the reciprocal loss of expression of CK 19 and the reappearance of CK 18 as malignant tumors developed. Invasive cells also did not express CK 13. Our results, based on the rat model, are similar to those reported by others on CK expression in human bladder tumors. Because these changes in CK expression occurred at specific points in the progression of urothelial lesions, the antibodies utilized in this study may be helpful in predicting the invasive potential of cells present in cytopathological specimens and tissue biopsies from human urothelial lesions.

[Carcinosarcoma and spindle cell carcinoma of the bladder. A comparison of 2 cases with an immunohistochemical study]. / Carcinosarcome et carcinome à cellules fusiformes de la vessie. Comparaison de deux observations avec étude immunohistochimique.

These two cases of bladder tumours with an unusual histological appearance were observed at Hôpital Saint-Louis in 1988. They contained two cellular components: the usual epithelial type and a spindle cell type. Immunohistochemistry performed in order to identify the various cell contingents established the diagnoses of carcinosarcoma and spindle cell carcinoma and emphasised the differences and similarities between these two entities, which we believe can be differentiated.

Carcinoma of the prostate simulating primary bladder cancer.

After encountering two patients who were diagnosed as having primary bladder cancer but were ultimately found to have carcinoma of the prostate invading the bladder, a prospective study was undertaken. In five patients with known carcinoma of the prostate invading the bladder, cup biopsies of the bladder lesions were reported as primary bladder cancer in four patients. To prevent this error, immunohistochemical studies are required and discussed in this paper.

Comparative flow cytometric deoxyribonucleic acid studies on exophytic tumor and random mucosal biopsies in untreated carcinoma of the bladder.

In 290 patients with untreated carcinoma of the bladder the deoxyribonucleic acid properties, as measured by flow cytometry, of 3 random mucosal biopsies were studied and compared to those of the exophytic tumors. Mucosal aneuploidy was found with few exceptions in aneuploid tumors only, and in a significantly lower frequency in aneuploid tumors of grade 2 than grade 3. The individual specificity of bladder tumors is emphasized by the observation that the level of ploidy was mostly the same in aneuploid mucosal biopsies as in the exophytic tumor. This is underlined further by the occurrence of cell populations of the same ploidy in different parts of the bladder mucosa. However, S-phase values of the concomitant intraurothelial lesions were significantly lower than those of the exophytic tumors. Therefore, we concluded that the process of evolution from malignantly transformed lesions, confined to the urothelium, to an exophytic or invasive tumor is dependent on a further elevated proliferation of the urothelial lesions.

Endometriose vesical / Endometriosis of bladder

Os autores apresentam 01 caso de endometriose vesical em paciente do sexo feminino, de 42 anos de idade, que apresentava quadro de dor abdominal e disúria importante. Foi tratada com sucesso por ressecçäo endoscópica vesical, histerectomia e ooforectomia.

Detection of bladder carcinoma in females by flow cytometry and cytology.

The sensitivity of bladder wash flow cytometry (BWFCM), voided urinary cytology (VUC), and cytology of catheterized urine obtained at the time of cystoscopy (CUC) were reviewed on all women evaluated for bladder cancer at Memorial Sloan-Kettering Cancer Center between June 1985 and December 1986. This comprised sixty-four episodes of pathologically proven bladder cancer in 48 women. Considering positive and suspicious results jointly the sensitivities of BWFCM, CUC and 3 VUC were 75%, 64% and 56%, respectively. If only positive results were considered (i.e., suspicious results considered as negative), the sensitivities of BWFCM, CUC and 3 VUC were 64%, 31% and 32%, respectively. The sensitivities of these tests are less than for a predominantly male population, presumably related to the presence of squamous epithelium and greater frequency of pyuria. However, bladder wash flow cytometry and conventional cytology are still a very valuable addition to cystoscopic examination, and the combination of BWFCM with conventional cytology is more sensitive than either procedure alone.

Measurement variability in DNA flow cytometry of replicate samples.

A Bladder Cancer Flow Cytometry Network study has been carried out aimed at identification of the sources of inter- and intralaboratory variability. Replicate "cocktail" samples containing a mixture of peripheral blood lymphocytes and an aneuploid cell line and samples of peripheral blood lymphocytes serving as a DNA reference standard were distributed to five network laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Sets of these samples were analyzed by flow cytometry to obtain cellular DNA distributions. DNA index and hyperdiploid fraction were calculated for each histogram using an automated technique. Results were evaluated by analysis of variance to identify sources of variability. Three important sources of variation were found that affect flow cytometry in general and- the transportability of flow cytometry results to routine clinical use in particular. The significant variation among laboratories that is constant across time most probably represents stable differences in instrumentation, instrument set-up, and laboratory techniques. This variation can be compensated for, if it is known and stable, to develop transportable classification criteria. The second type of variation, termed the interaction component, represents differences among laboratories that are not constant across time. Sources of this variation include inconsistency in sample preparation, staining, and analysis. The elimination of this type of variation is required for meaningful comparison of data within and among laboratories and the creation of interlaboratory data-bases. The third type of variation represents pure measurement variability and affects the sensitivity of the technique.