DNA study of bladder papillary tumours chemically induced by N-butyl-N-(4-hydroxybutyl) nitrosamine in Fisher rats

To examine DNA abnormalities in bladder papillary tumours induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in female rats, using image cytometric DNA analysis and cytogenetics. Thirty female rats were exposed to BBN in their drinking water for 20 weeks. One group of 10 animals served as controls. The animals exposed to BBN were killed at a rate of two per week, with the bladder being collected under aseptic conditions and those tumours with exophytic growth removed. The nuclear DNA content of the tumours was evaluated using image cytometric analysis. In two rats part of the tumour pieces was stipulated for culturing. Cytogenetic analysis was performed on at least 30 cells from each cell population and on both tumours. Papillary carcinomas were classified as low grade and high grade. DNA ploidy studies were carried out on 28 low-grade and 21 high-grade papillary carcinomas. Histograms obtained by image analysis showed that a normal urothelium was diploid; 28.6% and 100% of low-and high-grade papillary carcinomas were aneuploid respectively. Both tumours used for cell culture showed multiple numerical and structural chromosome alterations and several marker chromosomes. Image cytometric DNA analysis proved to be a good and reliable method for examining DNA alterations in papillary bladder carcinomas. The present findings establish that the DNA content is statistically different between low-grade and high-grade papillary carcinomas and that deviation from the diploid number is markedly higher in the high-grade ones. In addition, the occurrence of marker chromosomes seems to be related to the aggressiveness of the tumour.     

Prognostic factors in invasive cervical carcinomas associated with human papillomavirus (HPV). Quantitative data and cytokeratin expression.

As a part of a larger programme to search for the prognostic factors in cervical cancer, quantitative morphometry, demonstration of AgNORs and expression of different cytokeratin polypeptides (SK2-27, SK1, A 53-B/A2) were used to study a series of 85 cervical squamous cell carcinomas, previously analysed for the presence of human papillomavirus (HPV) DNA by in situ hybridization and polymerase chain reaction (PCR). The following nuclear profile parameters were calculated: nuclear area, perimeter, maximum diameter, ellipsoidity (form Ell), regularity (form Ar) and roundness (form Pe). In each case, the number of small (< 3 microns), large (> 3 microns), the total number and the ratio large/small AgNORs were registered. The cancer cell density and the lymphoid cell density were assessed. In the survival analysis, neither the expression of different cytokeratin polypeptides or the pattern of cytokeratin staining proved to be an independent variable. Similarly, none of the nuclear profile parameters analysed possessed an independent prognostic value in the survival analysis. The ratio of large/small AgNORs proved to be a significant independent prognostic predictor (p = 0.0104), second only to the lymphoid cell density. Also the total number of AgNORs was a prognostic indicator. This suggests that AgNOR size and ratio reflect tumor proliferation also in cervical squamous cell carcinoma, as shown in other human malignancies. Similarly, the density of cancer cell nuclei proved to be an independent prognostic predictor (p = 0.0601) in that the tumours in patients with longer survival showed lower density of the nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of uroplakin expression during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rats and mice

The expression of uroplakins, the tissue-specific and differentiation-dependent membrane proteins of the urothelium, was analyzed immunohistochemically in N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-treated rats and mice during bladder carcinogenesis. Male Fischer 344 rats were treated with 0.05% BBN in the drinking water for 10 wk and were euthanatized at week 20 of the experiment. BBN was administered to male B6D2F, mice; it was either provided at a rate of 0.05% in the drinking water (for 26 wk) or 5 mg BBN was administered by intragastric gavage twice weekly for 10 wk, followed by 20 wk without treatment. In rats, BBN-induced, noninvasive, low-grade, papillary, transitional cell carcinoma (TCC) showed decreased uroplakin-staining of cells lining the lumen but showed increased expression in some nonluminal cells. In mice, nonpapillary, high-grade dysplasia, carcinoma in situ, and invasive carcinoma were induced. There was a marked decrease in the number of uroplakin-positive cells lining the lumen and in nonluminal cells. This occurred in normal-appearing urothelium in BBN-treated mice and in dysplasic urothelium, in carcinoma in situ, and in invasive TCC. The percentage of uroplakin-positive nonluminal cells was higher in control mice than in rats, but it was lower in the mouse than in the rat after BBN treatment. Uroplakin expression was disorderly and focal in BBN-treated urothelium in both species. These results indicate that BBN treatment changed the expression of uroplakins during bladder carcinogenesis, with differences in rats and mice being related to degree of tumor differentiation.     

膀胱移行细胞癌中血型抗原Lewis A和Lewis X表达

目的　探讨血型抗原LewisA和LewisX在膀胱移行细胞癌中的表达及其诊断应用价值。方法　采用免疫组织化学EnVision方法 ,测定血型抗原LewisA和LewisX在 83例膀胱移行细胞癌和 6 8例非肿瘤性移行上皮黏膜中的表达 ,并收集10例膀胱癌及 10例正常人的尿脱落细胞标本进行LewisX免疫染色。结果　LewisA和LewisX在膀胱癌中的表达阳性率分别为 81 9% (6 8/ 83)和 83 1% (6 9/ 83) ,非肿瘤黏膜中的表达阳性率分别为 5 2 9% (36 / 6 8)和 11 8% (8/ 6 8)。LewisA和LewisX的表达强度与膀胱癌的病理分级和临床分期无关 (P >0 0 5 ) ,但膀胱癌的LewisA和LewisX表达强度高于非肿瘤黏膜 (P <0 0 5 )。 2 0例尿脱落细胞标本中 ,8例膀胱癌LewisX表达阳性 ,正常人组全部阴性。结论　LewisA和LewisX可成为诊断膀胱移行细胞癌的参考指标 ,特别是LewisX ,有助于低级别移行细胞癌的诊断     

膀胱移行细胞癌细胞周期调节相关基因表达与肿瘤生物学行为的关系

目的：探讨细胞周期调节相关基因p16INK4,p21WAF／CIP1,p53mlt在膀胱移行细胞癌中的表达与肿瘤增殖能力,病理分级及临床分期的关系。方法：应用免疫组织化学技术分析77例膀胱移行细胞癌组织p16INK4,p21WAF/CIP1,p53mlt基因的表达和增殖细胞核抗原（PCNA）表达情况,并与病理分级及临床分期之间进行综合分析。结果：p16INK4,p21WAF/CIP1,p53mlt在膀胱移行细胞癌中的表达及PCNA增殖指数与肿瘤的病理分级有关,与临床分期无关。p16,p21,p53阳性组与阴性组分别比较,其PCNA增殖数之间有差异性。多因素分析发现,p16INK4和p21WAF／CIP1阴性及p53mlt阳性组的PCNA值明显高于p16INK4和p21WAF／CIP1阳性及p53mlt阳性组,两者比较不同病理分级的阳性表达构成比亦有显著性差异。结论:联合检测p16INK4,p21WAF/CIP1,p53mlt基因的表达情况能充分反映膀胱移行细胞癌的增殖能力及生物学行为,对膀胱移行细胞癌患者的预后判断及治疗有指导意义。     

Distribution of cytokeratin polypeptides in human transitional cell carcinomas, with special emphasis on changing expression patterns during tumor progression.

The expression of cytokeratin (CK) polypeptides was studied in 59 transitional cell carcinomas (TCC) of the urinary tract of different grade and stage. Using a panel of 14 polypeptide-specific monoclonal CK-antibodies we identified immunohistochemically 8 different CKs separately, ie, CKs 4, 7, 8, 10, 13, 14, 18, and 19, while in immunoblotting studies CK5 expression was detected indirectly by using the antibody RCK102, recognizing CK5 + 8. In low-grade TCCs (G1-G2), the CK distribution was comparable to that in normal urothelium, however with a variable expression of CK13 in the different tumors and a uniform distribution of CK7. In higher-grade TCCs (G3), a decrease in CK13 expression was observed, particularly in the areas of muscle invasion. Furthermore, the appearance and increasing expression of CK14 (not present in normal urothelium or G1 TCCs) with higher grade and stage was striking. With tumor progression changes in epitope configurations of CK8 and CK18 were detected, as concluded from immunohistochemical assays with the panel of monoclonal antibodies for each of these two CKs. In extreme cases this resulted in differential staining patterns of the invasive and noninvasive components within one tumor. In 7 of 32 G3 TCCs, some of which showed areas with evident squamous differentiation, a decrease in the expression of CK7 and/or CK8 was seen. We conclude that tumor progression in TCCs is associated with discrete changes of CK expression, which can be detected using monoclonal antibodies.     

Cytokeratins in normal and malignant transitional epithelium. Maintenance of expression of urothelial differentiation features in transitional cell carcinomas and bladder carcinoma cell culture lines.

The pattern of cytokeratins expressed in normal urothelium has been compared with that of various forms of transitional cell carcinomas (TCCs; 21 cases) and cultured bladder carcinoma cell lines, using immunolocalization and gel electrophoretic techniques. In normal urothelium, all simple-epithelium-type cytokeratins (polypeptides 7, 8, 18, 19) were detected in all cell layers, whereas antibodies to cytokeratins typical for stratified epithelia reacted with certain basal cells only or, in the case of cytokeratin 13, with cells of the basal and intermediate layers. This pattern was essentially maintained in low-grade (G1, G1/2) TCCs but was remarkably modified in G2 TCCs. In G3 TCCs simple-epithelial cytokeratins were predominant whereas the amounts of component 13 were greatly reduced. Squamous metaplasia was accompanied generally by increased or new expression of some stratified-epithelial cytokeratins. The cytokeratin patterns of cell culture lines RT-112 and RT-4 resembled those of G1 and G2 TCCs, whereas cell line T-24 was comparable to G3 carcinomas. The cell line EJ showed a markedly different pattern. The results indicate that, in the cell layers of the urothelium, the synthesis of stratification-related cytokeratins such as component 13 is inversely oriented compared with that in other stratified epithelia where these proteins are suprabasally expressed, that TCCs retain certain intrinsic cytoskeletal features of urothelium, and that different TCCs can be distinguished by their cytokeratin patterns. The potential value of these observations in histopathologic and cytologic diagnoses is discussed.     

Nonspecific esterase reaction in hyperplastic urinary bladder epithelium induced by administration of N-butyl-N-(4-hydroxybutyl)nitrosamine,freezing and formalin instillation in rats.

Chronological changes in nonspecific esterase (NSE) activity in hyperplasia of the bladder mucosa in Wistar rats induced by the administration of 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in their drinking water for up to 20 weeks and in reversible regenerative hyperplasia by freeze ulceration and 20% formalin instillation in the bladder were compared. In regenerative hyperplasia foci with strong NSE activity could not be proved throughout the experimental period, while the foci were detected in hyperplastic epithelium induced by BBN treatment for more than 3 weeks. The focus of NSE high activity persisted for 56 weeks after withdrawal of the carcinogen and the focus or area with the same NSE reaction appeared in papilloma and transitional cell carcinoma seen in weeks 7 to 20 of BBN treatment. The appearance of focal strong activity of NSE seemed to be a promising marker for the precursor lesions of bladder tumors. Short uniform, pleomorphic microvilli were observed on the cell surface of preneoplastic and carcinomatous lesions by BBN as well as on that of regenerative hyperplasia after freeze ulceration and formalin instillation.     

Microcystic transitional cell carcinomas of the urinary bladder. A report of four cases

Four transitional cell carcinomas of the urinary bladder that on microscopic examination were found to have cysts are described. The tumors occurred in patients from 35 to 69 years of age and were all deeply invasive; two were grade 2 and two grade 3 (of three grades). Cysts were prominent in all of the cases and predominated in one of them. In the latter case, the cysts caused major problems in interpretation. The cysts, which usually were round to oval and were as large as 1.2 mm, were of varying sizes. They usually were lined by transitional cells or low columnar cells showing mucinous differentiation; occasionally they were lined by a single layer of flattened cells or the lining epithelium was denuded. Elongated, irregular branching spaces also were present. Recognition of these tumors is facilitated by their association with typical transitional cell carcinoma, but when the cysts predominate, awareness of this change may be essential in establishing the diagnosis, particularly in a biopsy specimen.     

Immunohistochemically demonstrated variation in expression of cathepsin E between uracil-induced papillomatosis and N-butyl-N-(4-hydroxybutyl)nitrosamine-induced preneoplastic and neoplastic changes in rat urinary bladder

Expression of rat urinary bladder cathepsin E in benign papillomatosis induced by uracil and various stages of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced carcinogenesis was investigated immunohistochemically. Seven-week-old, male F344/DuCrj rats were used. In the normal urothelium of control rats, cathepsin E stained in all layers of cells, although in umbrella cells and some basal cells the reaction was relatively weak. In rats given a diet containing 3% uracil for 5 weeks immunoreactivity of cathepsin E in uracil-induced papillomatosis was consistently homogeneous in all layers, but weaker than in normal urothelium. In rats given 0.05% BBN in drinking water for 12 weeks and subsequently maintained without treatment for 48 weeks cells with little cathepsin E, never observed in normal urothelium, appeared at 5 weeks above the basement membrane in the earliest stage of BBN-induced urinary bladder cancer (simple hyperplasia). Throughout the neoplastic process, groups of cells with a little cathepsin E were randomly distributed, with expression in the urothelium being markedly unstable. Almost all areas of squamous cell proliferation in TCC were negative for cathepsin E. Instability of cathepsin E expression in rat urothelium therefore appears characteristic for carcinogenesis and offers the possibility of using this feature as an early biomarker for urinary bladder carcinogenesis.     

Reciprocal correlation between the expression of cyclooxygenase-2 and E-cadherin in human bladder transitional cell carcinomas

Carcinoma cells become more motile and invasive via downmodulation of E-cadherin. Cyclooxygenase-2 (COX-2) expression is associated with tumor invasion and metastasis. The aim of this study is to investigate the relationship between the expression of COX-2 and E-cadherin in a bladder cancer cell line and human bladder transitional cell carcinoma (TCCs). Phorbol 12-myristate 13-acetate (PMA) treatment for 5637 bladder cancer cells increased COX-2 expression, slightly induced Slug expression, and decreased E-cadherin expression. Ectopic expression of COX-2 or prostaglandin E₂ (PGE₂) treatment for 5637 cells reduced E-cadherin expression. This finding was confirmed by the result that knockdown of COX-2 expression or indomethacin administration increased the expression of E-cadherin. When compared with cells’ motility in serum-free medium, the treatment of PMA and PGE₂ increased cell motility, and indomethacin treatment slightly decreased cell motility. In the tissues of bladder TCCs, COX-2 expression was inversely correlated with membranous E-cadherin expression and positively correlated with nuclear β-catenin expression. The expression of COX-2 and nuclear β-catenin expression was significantly higher in TCCs of high grade and invasive growth than in TCCs of low grade and noninvasive growth. In contrast, membranous E-cadherin expression was more decreased in tumors of high grade and invasive growth. In addition, nuclear β-catenin expression was significantly related to tumor recurrence. We suggest that COX-2 pathway reduces membranous E-cadherin expression in bladder TCCs and their expression pattern may provide important information in predicting the clinical behavior of bladder TCCs.     

Quantitative interrelations of Lewis antigens in normal mucosa and transitional cell bladder carcinomas.

The factors regulating the expression of the Lewis blood group related antigens in tissues have yet to be clarified. In an attempt to resolve some of the existing controversies the quantitative interrelationship of the Le(a), Le(b), X and Y antigens in normal urothelium and transitional cell carcinomas (TCC) was studied using biopsy specimens derived from 22 patients whose ABO and Lewis red blood cell phenotype was known. A quantitative scale was devised to encompass both the extent and intensity of the immunohistochemical reactivity in one numerical value (score). The expression of these four antigens in the normal urothelium followed a characteristic pattern that is related to but not identical with the red blood cell phenotype. An excess of Le(b) and Y in the urothelium correlated with the Le(a-b+) red blood cell phenotype, while a relative increase in Le(a) and X (at the expense of Le(b) and Y) was associated with the Le(a+b-) red blood cell phenotype. This pattern can be accounted for by the combined effects of differential gene expression and substrate availability. The quantitative comparison of the antigenic make-up of TCCs with the corresponding normal tissue phenotype shows consistent trends, suggesting that the changes associated with neoplasia derive primarily from the suppression of specific gene products and, secondarily, from altered competitive substrate utilisation.     

Nonspecific esterase reaction in hyperplastic urinary bladder epithelium induced by administration of N-butyl-N-(4-hydroxybutyl)nitrosamine, freezing and formalin instillation in rats

Chronological changes in nonspecific esterase (NSE) activity in hyperplasia of the bladder mucosa in Wistar rats induced by the administration of 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in their drinking water for up to 20 weeks and in reversible regenerative hyperplasia by freeze ulceration and 20% formalin instillation in the bladder were compared. In regenerative hyperplasia foci with strong NSE activity could not be proved throughout the experimental period, while the foci were detected in hyperplastic epithelium induced by BBN treatment for more than 3 weeks. The focus of NSE high activity persisted for 56 weeks after withdrawal of the carcinogen and the focus or area with the same NSE reaction appeared in papilloma and transitional cell carcinoma seen in weeks 7 to 20 of BBN treatment. The appearance of focal strong activity of NSE seemed to be a promising marker for the precursor lesions of bladder tumors. Short uniform, pleomorphic microvilli were observed on the cell surface of preneoplastic and carcinomatous lesions by BBN as well as on that of regenerative hyperplasia after freeze ulceration and formalin instillation.     

Cytokine-induced gene expression of interleukin-8 in human transitional cell carcinomas and renal cell carcinomas.

Chemotactic cytokines play a critical role in recruiting leukocytes to sites of tissue injury. Interleukin-8 (IL-8) is a chemotactic cytokine secreted by a variety of cells (eg, monocytes, endothelial cells, fibroblasts) during the inflammatory response. In this report, the authors demonstrate that human transitional cell carcinomas and renal cell carcinomas have the capacity to elaborate IL-8 in response to the inflammatory mediators IL-1 beta and tumor necrosis factor (TNF)-alpha. All cell lines expressed high levels of IL-8 mRNA on stimulation with either IL-1 beta or TNF-alpha, but not lipopolysaccharide; one expressed the gene constitutively. The authors selected one transitional cell carcinoma cell line (UM-UC-9) and one renal cell carcinoma cell line (UM-RC-5) for further study. Both displayed a time- and dose-dependent increase in steady-state levels of IL-8 mRNA in response to IL-1 beta and TNF-alpha. Specific mRNA was detectable by 1 hour after stimulation. Secretion of antigenic IL-8 measured by enzyme-linked immunosorbent assay into culture supernatants reflected the kinetics of mRNA expression. Because heat-inactivated TNF-alpha failed to induce synthesis of IL-8 mRNA, and cycloheximide augmented TNF-alpha-induced synthesis, IL-8 expression appears to be a stimulus-specific primary induction phenomenon. As with other inflammatory mediators whose mRNA contains a 3' AU-rich sequence (eg, IL-2, TNF-alpha), the half-life of IL-8 mRNA was short, less than 1 hour. Our data suggest that secretion of IL-8 by malignant cells may partly account for the inflammatory infiltrates associated with some malignant neoplasms.     

Comparison of cytokeratin expression in primary and metastatic carcinomas. Diagnostic application in surgical pathology

Metastatic poorly differentiated carcinomas often represent diagnostic difficulties in surgical pathology. Therefore, the expression of cytokeratins of different molecular weights (54, 57, and 66 kd) were compared in paraffin sections of 37 primary carcinomas with their lymph node metastases by an avidin-biotin complex (ABC) method, using monoclonal antibodies. The epithelial tumors consisted of 16 squamous cell carcinomas (SCCs) and 17 adenocarcinomas with different degrees of differentiation (well, moderately, or poorly differentiated), a renal cell carcinoma, a hepatocellular carcinoma, a transitional cell carcinoma of the bladder, and a carcinoid tumor of the stomach. The primary and metastatic tumors showed the same cytokeratin profiles. All SCCs and their metastases were positive for 57-kd cytokeratin and negative for 54-kd cytokeratin. All adenocarcinomas and their metastases were positive for 54-kd cytokeratin and negative for 66-kd cytokeratin. The extent of reactions varied with the differentiation of the carcinomas, with well-differentiated tumors showing more diffuse staining. Cases of lymphoma, sarcoma, and melanoma were negative for the three types of cytokeratins. The results indicate that identification of different molecular weight cytokeratins may be used to distinguish poorly differentiated SCCs from poorly differentiated adenocarcinomas, even in metastatic tumors. In addition, demonstration of these cytokeratins is useful in substantiating presence and identity of small foci of metastases in lymph nodes.     

Altered expression of UPIa,UPIb, UPII,and UPIIIa during urothelial carcinogenesis induced by N-butyl-N-(4-hydroxybutyl)nitrosamine in rats

In normal urothelium, superficial umbrella cells express four major integral membrane proteins, uroplakins UPIa, UPIb, UPII, and UPIIIa, which compose urothelial plaques. In the apical plasma membrane, urothelial plaques form microridges. During neoplastic changes, microridges are replaced by microvilli, while uroplakin expression is retained. We correlated individual uroplakin expression with apical plasma membrane structure, cytokeratin 20 expression, and urothelial cell proliferation (Ki-67). Male Wistar rats were treated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water, which caused flat hyperplasia with mild dysplasia, low-grade papillary urothelial carcinoma, invasive low- and high-grade papillary urothelial carcinoma and invasive squamous cell carcinoma with extensive keratinization, grade 2. During urothelial carcinogenesis, UPII expression was the most decreased in all urothelial lesions, while UPIa, UPIb, and UPIIIa expression was differently altered in different types of lesions. Superficial cells were covered with microvilli and ropy ridges, while microridges were disappearing. The expression of cytokeratin 20 was decreased and limited to superficial urothelial cells. Proliferation indices were increased, except for invasive squamous cell carcinoma with extensive keratinization. Our results indicate that during urothelial carcinogenesis the expression of UPII is diminished, suggesting that UPIb/UPIIIa heterodimer can still be formed, while heterodimer UPIa/UPII formation is disrupted. Correlation between decreased level of UPII expression and changed apical plasma membrane structure suggests that diminished expression of UPII hinders the urothelial plaque formation.     

Improved objectivity of grading of T(A,1) transitional cell carcinomas of the urinary bladder by quantitative nuclear and proliferation related features.

AIM: To analyse whether the mean nuclear area of the 10 largest nuclei (MNA-10), the mitotic activity index (MAI), and Ki-67 immunoquantitative features have additional value to discriminate different grades of T(A,1) transitional cell carcinoma (TCC) of the urinary bladder. MATERIALS/METHODS: One hundred and fifty of 200 consecutive cases (75%) showing interobserver agreement on duplicate blind grade assessment by independent pathologists were studied. Using random numbers, the 150 cases were divided into sets for learning (n = 75) and testing (n = 75). Single and multivariate analyses were applied to discriminate the different grades in the learning set. The multivariate classifier developed in this way was evaluated in the test set (n = 75). RESULTS: With the MNA-10 alone, using the classification MNA-10 < 80 microm(2) = grade 1, 80 microm(2) < MNA-10 < 130 microm(2) = grade 2, MNA-10 > 130 microm(2) = grade 3, 71% of all 150 cases were correctly classified (69% of grade 1 v grade 2 and 76% of grade 2 v grade 3). With multivariate analysis, the best discriminating features in the learning set (17 grade 1, 30 grade 2, and 28 grade 3) between grades 1 and 2 were MNA-10 and MAI, and between grades 2 and 3 MAI and Ki-67. With these features, 94% of grade 1 v grade 2 and 97% of grade 2 v grade 3 were correctly classified in the learning set (overall, 95% correct, none of the grade 3 cases misclassified). In the test set the classification results were similar. When the three grades were entered at the same time for discrimination, Ki-67 area % and MAI was the best discriminating combination, both in the sets for learning and testing. Overall correct classification results in the sets for learning and testing were slightly lower, but still 94% and 92%. Most importantly, none of the grade 3 cases was misclassified; the classification shifts all occurred between grades 1 and 2. CONCLUSIONS: The combination of MNA-10, MAI, and Ki-67 gives much better discrimination between grades 1, 2, and 3 in T(A,1) TCC of the urinary bladder than MNA-10 alone. The similarity of the classification results of the learning set and test set are encouraging and this quantitative pathological grading model should be applied in a prospective study.     

Specific genomic alterations in rat renal cell carcinomas induced by N-ethyl-N-hydroxyethylnitrosamine.

To characterize genetic alterations occurring in renal tumorigenesis, EHEN-induced renal cell tumors were examined using restriction landmark genomic scanning (RLGS) analysis, an electrophoretic separation technique that detects gene amplifications and deletions. Comparison of DNAs from tumor against those from corresponding nontumorous kidney and/or EHEN-treated kidney without development of renal tumors yielded specific alterations in terms of both amplified and reduced DNA spots. Two amplified spots were detected only in renal cell tumors and an additional four spots were frequent in EHEN-treated kidneys. One reduced spot was common to all tumor samples, and another was frequently detected in the tumors analyzed but not in EHEN-treated kidneys. A subset of the altered spots thus appeared to be specific for EHEN-induced renal tumorigenesis.     

Correlation of DNA ploidy levels with altered cytokeratin patterns in rat bladder tumors.

Flow cytometry analysis of primary bladder carcinoma cells is appropriate for demonstrating the association between the presence of aneuploid cells and increased malignant potential for transitional cell carcinoma (TCC). Our laboratory previously has reported a specific alteration in the cytokeratin (CK) pattern of experimentally induced bladder carcinomas in rats. Unique immunohistochemically detectable changes in CK expression were the loss of CK 13 expression in invasive TCC cells and the loss of CK 19 expression in induced TCC. We now report the correlation of these changes in CK expression with cellular aneuploidy of the induced bladder carcinomas. Bladder carcinomas were induced in rats by direct instillation of N-methyl-N-nitrosourea. Immunohistochemistry was performed on the induced tumors using four commercially available monoclonal antibodies specific for CK 18 (TR1031), CK 13 (K8.12), CK 19 (K4.62), and the CKs 5, 7, and 8 (K8.13). Flow cytometry data from the induced bladder carcinomas was analyzed and the DNA index, proliferative index, and percent aneuploid cells were calculated for each time point. The percent aneuploid cells and CK 19 staining were tested statistically and were shown to be negatively correlated. We therefore hypothesize that the combination of the loss of CK 19 as detected by the antibody K4.62 coupled with the presence of aneuploid cells in histopathologically diagnosed invasive TCC is a significant factor in predicting the prognosis for any given diagnosis.