Activation of H-ras oncogene in rat bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine.

Ras-oncogene activation was investigated in the bladder tumors of F344 male rats given N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water. DNA from one of the nine transitional cell carcinomas contained an H-ras oncogene detectable by the NIH/3T3 transfection assay. Analysis of p21 ras proteins suggested that the activating mutation resided within codon 61 of the H-ras gene and that such activating mutations were not present in other tumors. In contrast to mutational activation of ras genes, enhanced expression of p21 was observed in all tumors examined by immunohistochemical techniques with the use of Formalin-fixed paraffin-embedded tissue sections and an anti-ras p21 antibody, RAP-5. Further histochemical analysis of bladder tissues at various stages of the BBN-induced carcinogenic process indicated that the enhanced expression of p21 appeared early; the reactivity with RAP-5 was observed in diffuse hyperplastic epithelia after 5 weeks of exposure to BBN. The frequency of ras oncogenes, activated either by point mutations or overexpression of p21, in BBN-induced rat bladder carcinomas has thus been shown to be similar to that observed in human bladder carcinomas.

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.

Small cell neuroendocrine carcinoma of the colon and rectum: clinical, histologic, and ultrastructural study and immunohistochemical comparison with cloacogenic carcinoma.

In an effort to provide immunocytochemical data that would be useful in distinguishing between small cell epithelial tumors of the anorectal region, 10 cases of neuroendocrine small cell colorectal carcinoma (NSCCC) and five cases of cloacogenic carcinoma (CC) were studied with antibodies to cytokeratin, epithelial membrane antigen (EMA), chromogranin, blood group isoantigens (BGI), carcinoembryonic antigen (CEA), Leu-M1, Leu-7, leukocyte common antigen (LCA), S-100 protein, neurofilaments (NF), neuron-specific enolase (NSE), serotonin, and 14 neuropeptides. The diagnoses for all 15 tumors were verified ultrastructurally. Among the antigenic determinants considered, reactivity for low- and medium-molecular-weight cytokeratin, EMA, NSE, and NF was seen in the majority of NSCCCs, whereas the CCs were positive for all cytokeratin classes, BGI, EMA, and CEA. In addition, Leu-M1, Leu-7, and chromogranin were each expressed in three cases of NSCCC. None of the other antisera yielded positive results in tumors of either type. All 10 patients with NSCCC died of their tumors within 11 months of clinical presentation, while four of the five CCs proved fatal, with an average survival of 28 months. One of the patients with CC was free of disease 31 months after diagnosis. These data suggest that an immunocytochemical panel, consisting of antibodies to high-molecular-weight cytokeratin, BGI, CEA, NSE, and NF (and possibly Leu-7 and chromogranin as well), is capable of distinguishing between NSCCC and CC in problematic cases. Although tumors of both types are aggressive, it is possible that the survival statistics for both may be improved through more accurate diagnostic classification.

Immunohistochemical localization of keratin-type proteins in epithelial neoplasms. Correlation with electron microscopic findings.

A rabbit antiserum prepared against human keratins isolated from calluses was applied to sections of 108 neoplasms using indirect immunofluorescence and immunoperoxidase technics. The vast majority of epithelial neoplasms were strongly positive for keratin-type proteins, even in the absence of obvious keratinization or squamous differentiation as revealed by light microscopy. This keratin-positivity was invariably correlated with the identification of intermediate-sized filaments arranged in loose or dense bundles in the cytoplasm of neoplastic epithelial cells. Keratin-negative neoplasms included nevi, malignant melanomas, carcinoid tumors, malignant lymphomas, and a variety of connective-tissue tumors. Immunologic identification of keratin-type proteins was particularly helpful in establishing the epithelial nature of "undifferentiated" malignant tumors, including oat cell carcinomas.

Immunohistochemistry of cytokeratin proteins in squamous and transitional cell lesions of the urinary tract.

Expression of low and high molecular weight cytokeratin proteins was investigated immunohistochemically in a variety of transitional and squamous epithelial lesions of the urinary tract with and without schistosomiasis. The monoclonal antibodies used were CAM 5.2 and NCL5D3 for low, PK 63 and 121 for high, and MAK 6 for a broad range of intermediate molecular weight cytokeratins. On staining with CAM 5.2 and NCL5D3, urothelial hyperplasias (n = 12) and grades 1 (n = 5) and 2 (n = 10) papillary transitional cell carcinomas showed labelling patterns quite distinct from carcinoma in situ (n = 4) and non-papillary grades 2 (n = 6) and 3 tumours (n = 3). Among squamous lesions only focal positivity was obtained in 14 of 22 moderate to poorly differentiated squamous cell carcinomas. By contrast, PK 63 and 121 stained squamous lesions exclusively. MAK 6 stained the whole range of urothelial and squamous lesions with the exception of squamous metaplasias. Polyclonal antikeratin adequately labelled spindle cell areas of high grade tumours. The distinctive staining patterns given by these or similar antibodies may help in the identification of squamous metaplasia and in diagnosing tumours of the urothelium.

Retrospective study of prognostic importance of DNA flow cytometry of urinary bladder carcinoma.

Cellular DNA content was determined by flow cytometry on routinely processed paraffin sections of 61 primary and untreated transitional cell carcinomas of the urinary bladder, and correlated with tumour grade and stage and clinical follow up. All 16 (25%) grade 1 carcinomas were diploid and all 11 (20%) grade 3 tumours were aneuploid. The 34 (55%) grade 2 carcinomas comprised 13 (40%) diploid and 21 (60%) aneuploid cases. Among the 37 superficial carcinomas (stage Ta and T1), 25 (65%) were diploid; 20 (85%) of the 24 advanced tumours (stage T2 to T4) had aneuploid tracings. Ploidy was a significant prognostic indicator (p: 0.006) of five year survival. The initial presence of aneuploidy in superficial bladder carcinoma (stage Ta and T1) is a strong argument for more aggressive treatment than is customary.

Androgen receptors in bladder tumors.

Cytosol androgen receptor content of transitional cell bladder cancer tissue was found to be substantially higher than its content in normal bladder mucosa and lower than in control benign prostatic hypertrophy tissue. Tumors arising in female patients had a lower androgen receptor content than those arising in male patients. High-grade tumors had a lower androgen receptor content than low-grade tumors.

Sex hormone receptors in localized and advanced transitional cell carcinoma of urinary tract in humans.

Sex hormone receptors were identified in patients with localized and advanced transitional cell carcinoma of the bladder and kidney. Dihydrotestosterone and testosterone receptors were found most consistently in the specimens analyzed. Estrogen receptors were found less frequently. Androgen receptors in transitional cell carcinoma may prove to be a useful biologic marker in this disease.

Cytophotometric investigations of DNA-content in transitional cell tumors of the bladder. Comparison of results with clinical follow up.

Forty-nine patients with transitional cell tumors of the urinary bladder were followed up clinically for a period of at least 36 months (average time 61.4 months). Initial tumors were examined by means of DNA-Feulgencytophotometry. Histologic grading was performed according to Bergkvist et al. (1965). Clinical behavior clearly demonstrates the validity of the grading schedule used and shows the significance of DNA-Feulgencytophotometry for the standardization of bladder tumors and for the comparability of diagnostic and therapeutic results. Determination of the individual prognosis of low-malignancy tumors and the differentiation of histologic borderline cases does not appear to be possible by means of DNA-Feulgencytophotometry.

Keratin, luminal epithelial antigen and carcinoembryonic antigen in human urinary bladder carcinomas. An immunohistochemical study.

14 urinary bladder carcinomas of all main types were investigated with antisera to "broad spectrum keratin" (aK), "luminal epithelial antigen" (aLEA) and carcinoembryonic antigen (aCEA), using an indirect immunoperoxidase method on formalin fixed paraffin embedded sections. Keratin and LEA were both present in normal transitional epithelium, papilloma and carcinoma in situ whereas CEA was absent. Transitional cell carcinomas reacted with both aK and aLEA whereas CEA was seen only in a few foci. In squamous metaplasia and squamous carcinoma reaction with aK was particularly strong, while LEA was almost lacking and CEA was present in necrotic centres. In adenocarcinomas aK and aLEA reacted equally while aCEA reacted only on the surface.

Pituitary adenoma with multiple ciliated cysts: transitional cell tumor?

The case of a prolactin-secreting pituitary tumor with multiple cyst formation is described. Ultrastructurally, the cyst-lining cells were ciliated and closely resembled those of Rathke's cleft cyst but contained secretory granules, and no basal lamina formation was seen between the adenoma cells and the lining cells. Immunohistochemical study revealed that the adenoma cells consisted of both prolactin-secreting cells and growth hormone-secreting cells. The lining cells were immunoreactive with the antiserum to cytokeratin. No S-100 protein-positive cells were seen. The origin of this tumor is discussed.

[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.

[Chorionic gonadotrophic hormone and transitional cell carcinoma of the bladder. Immunohistochemical characterization of HCG and its alpha and beta subunits]. / Hormone gonadotrophine chorionique et carcinomes à cellules transitionnelles de la vessie. Caractérisation immunohistochimique de l'hCG et de ses sous-unités alpha et beta.

Investigation of cellular localization of hCG and alpha- and beta- subunits in 17 transitional cell carcinomas of urinary bladder was performed using immunohistochemical techniques. Serial sections were tested with monoclonal antibodies to hCG, free beta hCG and free alpha hCG. Transitional cell carcinomas producing hCG and/or beta hCG are easily found (6/17 cases). None of these tumors correspond morphologically to a choriocarcinoma. However, it must be mentioned that neoplastic areas exhibit some features reminiscent of a choriocarcinomatous structure. Notably, positive cells can be disposed at the periphery of neoplastic nests, mimicking the arrangement of syncytiotrophoblast. Positive neoplastic cells express hCG and/or beta hCG; alpha hCG immuno-reactivity is quite rare. The relative distribution of hCG/beta hCG is excessively variable from case to case. In contrast, normal transitional epithelium contains endocrine cells which only express the alpha-subunit of hCG.

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.

[Prognostic value of cytofluorometry in bladder tumors]. / Intérêt pronostique de la cytofluorométrie dans les tumeurs vésicales.

Bladder lavage fluid was examined using flow-cytometry (FCM) in 112 patients with transitional cell carcinoma seen over 30 months. FCM investigates the entire mucosa, furnishes indications as to the possible existence of dysplasia or carcinoma in situ, and thus provides for a more accurate evaluation of the evolutive potential of the "bladder disease". FCM consists in the automated assay of the DNA content of epithelial cells. The test is positive when "tumorous" diploid or aneuploid cells are demonstrated. The diagnostic sensitivity of FCM is comparable to that of cytologic diagnosis on bladder lavage specimens, but FCM has the additional advantage of detecting those patients at high risk for disease progression by measurement of the DNA index. Grade 1 and 2 tumors are diploid in 70% of patients, against only 14% for grade 3 tumors and carcinomas in situ. Follow up of 25 grade 2 patients and determination of the recurrence index clearly establishes the prognostic significance of the degree of tumorous ploidy. Furthermore, the effectiveness of endovesical chemotherapy can be monitored using FCM measurement of the aneuploidy index.

[Flow cytometric bromodeoxyuridine (BrdU)/DNA analysis--study with urogenital tumors].

We performed the simultaneous flow cytometric bromodeoxyuridine (BrdU)/DNA analysis in combination with in vitro BrdU labeling method using seven human urogenital tumors. The DNA content and incorporated BrdU content of each tumor cell was analyzed quantitatively using this flow cytometric method. The cell kinetics of each cell line of heterogeneous tumor could be analyzed by this combined method. In the near future, by establishing a procedure decreasing non-specific staining of cells, the development of this flow cytometric two-color analysis in clinical fields is expected.

[Studies on biological characteristics of transitional cell carcinoma of the bladder in patients under 30 years of age].

The incidence of transitional cell carcinoma (TCC) of the bladder has been increasing in men with a peak incidence occurring in the sixth decade. However, development of tumors under the age of 30 is relatively rare. In this regard, it has been reported that vesical tumors in the young group is less malignant and rare to recur when compared with those in the elderly group. Recently, flow cytometric DNA histograms (FCM) provides quantitative and objective informations for detection and evaluation of malignant potential of bladder neoplasms. Here we report patients with tumor of the bladder under 30 years old and assess the clinical properties and biological characteristics of their tumors based on FCM. A total of 11 patients from 1975 through 1988 were reviewed. Their mean age at the diagnosis was 22.6 years old (range from 22 to 29 years old). Male/female ratio was 2.7:1. The mean follow-up period was 4 years and 7 months (range from 8 months to over 13 years). An asymptomatic gross hematuria was found in all of the patients, which is the most common sign. Filling defects of the bladder on excretory urograms were observed in six out of the 11 patients (54.5%). Cystoscopically, the size of tumors was less than 2 cm in diameter. Ten patients had a single tumor and one patient had multiple tumors at the time of the initial diagnosis. Endoscopically tumors were papillary in all but one patient, who had a non-papillary tumor. Their urine cytology showed class I in one, class II in eight, class III in one, class IV in one and class V in none.(ABSTRACT TRUNCATED AT 250 WORDS)

[The DNA content of bladder carcinoma in relation to pathologic grading, staging and prognosis: a flow cytometric study].

DNA ploidy analysis of 90 paraffin embedded specimens in bladder carcinoma were carried out by flow cytometry (FCM). Emphasis was paid on grade II tumors which usually have variable clinical course. DNA ploid level determined by FCM correlates highly with pathologic grade, stage, and prognosis of bladder carcinoma. Among grade II tumors, the patients with diploid and peridiploid carcinoma had a favourable prognosis whereas the patients with aneuploidy of high average DNA index had a bad prognosis, in particular, those with triploid and peritriploid tumors had poorer prognosis. DNA ploid level by which the prognosis of patients with bladder carcinoma is evaluated, appears to be more accurate than pathologic grade and clinical stage, particularly stage T0-T or grade II tumors. It therefore can serve as a tumor marker in bladder carcinomas.

[Flow cytometry for the determination of DNA in head and neck carcinomas]. / La citometría de flujo para la determinación de DNA en los carcinomas de cabeza y cuello.

The technique used for DNA determination in tumoral cells from carcinomas of head and neck is described. Results obtained are commented on as is too the future of this technique for the better understanding of the behavior of the tumor and consequently of its treatment.

[Lectin receptors in transitional cell tumors of the bladder]. / Retseptory nekotorykh lektinov v perekhodno-kletochnykh opukholiakh mochevogo puzyria.

Binding sites for lectins of peanuts, soya-beans and concanavalin A were identified in the tissues of normal human urinary bladder and its transitional cell tumors (31 patients) using the lectin-peroxidase technique. No binding sites for any of the lectins were found in normal urothelium. Receptors for peanut and soya-bean lectins were observed on the plasma membranes of 20% of tumor cells in transitional cell papillomas. A diffuse distribution of those lectins in tumor cell cytoplasm was interpreted as a tendency to malignant transformation of tumor. There were no concanavalin A receptors in tumor cells.