Tumour recurrence has a major impact on patients with non-invasive papillary urothelial tumours of the bladder. To explore the role of DBC1 (deleted in bladder cancer 1 locus), a candidate tumour suppressor gene located at 9q32-33, as prognostic marker we have performed loss of heterozygosity (LOH) testing in 49 patients with primary papillary urothelial tumours and associated normal urothelium. Data from the 38 tumours and 11 specimens of normal urothelium that were informative in the LOH study (D9S195 marker) showed that LOH in urothelium (45.4%) but not in non-invasive tumours (60.5%) was associated with tumour recurrence (p = 0.026) but not to grade or progression. Also, tumours whose normal urothelium had LOH were larger (p = 0.020) and showed cyclin D1 over-expression (p = 0.032). Non-significant increased expression of p53, p21Waf1, apoptotic index and tumour proliferation, and decreased expression of p27Kip1 or cyclin D3 also characterized tumours whose normal urothelium had LOH. The expression of these G1-S modulators, apoptotic index and tumour proliferation was more heterogeneous in papillary urothelial tumours, irrespective of having retained heterozygosity or LOH. Also, Bax expression decreased in papillary urothelial tumours having LOH (p = 0.0473), but Bcl-2 was unrelated to LOH status. In addition, FGFR3 protein expression decreased in LOH tumours (p = 0.036) and in those having LOH in their normal urothelium (p = 0.022). FGFR3 immunohistochemical expression was validated by western blot in selected cases. The survival analysis selected LOH in normal urothelium as a marker of disease-free survival (log-rank 5.32, p = 0.021), progression-free survival (log-rank 3.97, p = 0.046) and overall survival (log-rank 4.26, p = 0.038); LOH in tumours was significant in progression-free survival (log-rank 3.83, p = 0.042). It is concluded that LOH at the DBC1 locus in normal urothelium seems to be relevant in the prognosis of non-invasive papillary tumours of the bladder via selecting cases with increased proliferation, frequent alterations of the G1-S phase modulators, and decreased FGFR3 protein expression.

Aims: To evaluate whether ki-67 labelling index (LI) has independent prognostic value for survival of patients with bladder urothelial tumours graded according to the 2004 World Health Organisation classification.Methods: Ki-67 LI was evaluated in 164 cases using the grid counting method. Non-invasive (stage Ta) tumours were: papilloma (n = 5), papillary urothelial neoplasia of low malignant potential (PUNLMP; n = 26), and low (LG; n = 34) or high grade (HG; n = 15) papillary urothelial carcinoma. Early invasive (stage T1) tumours were: LG (n = 58) and HG (n = 26) carcinoma. Statistical analysis included Fisher and chi(2) tests, and mean comparisons by ANOVA and t test. Univariate and multivariate survival analyses were performed according to the Kaplan-Meier method with log rank test and Cox's proportional hazard method.Results: Mean ki-67 LI increased from papilloma to PUNLMP, LG, and HG in stage Ta (p < 0.0001) and from LG to HG in stage T1 (p = 0.013) tumours. High tumour proliferation ( > 13%) was related to greater tumour size (p = 0.036), recurrence (p = 0.036), progression (p = 0.035), survival (p = 0.054), and high p53 accumulation (p = 0.015). Ki-67 LI and tumour size were independent predictors of disease free survival (DFS), but only ki-67 LI was related to progression free survival (PFS). Cancer specific overall survival (OS) was related to ki-67 LI, tumour size, and p27kip1 downregulation. Ki-67 LI was the main independent predictor of DFS (p = 0.0005), PFS (p = 0.0162), and cancer specific OS (p = 00195).Conclusion: Tumour proliferation measured by Ki-67 LI is related to tumour recurrence, stage progression, and is an independent predictor of DFS, PFS, and cancer specific OS in TaT1 bladder urothelial cell carcinoma.

Cyclin D3 deregulation has recently been reported in bladder cancer but its prognostic significance remains uncertain. A cohort of 159 patients with stage Ta or T1 primary bladder tumours was investigated to determine the significance of cyclin D3 expression in association with other G1-S phase regulators of the cell cycle (p53, p21Waf1, p27kip1, cyclin D1), including tumour proliferation (ki67-MIB1); its association with conventional clinicopathological parameters; and the relationship between cyclin D3 and loss of heterozygosity (LOH) at the 9p21 (p161NK4a locus) chromosome region. The end point of the study was progression-free survival. Cyclin D3, other G1-S phase regulators, and tumour proliferation were investigated by immunohistochemistry and measured by the grid-counting method. To validate the immunohistochemical expression, cyclin D3 was additionally assessed by western blotting in selected cases. LOH at the 9p21 chromosome region (marker D9S171) was assessed in 125 cases using an AB Prism 310 genetic analyser and a set of microsatellite fluorescence-la belled primers. Cyclin D3 overexpression was related to larger tumour size (> 5 cm; p < 0.0001) and high tumour proliferation (> 10%; p = 0.025). Mean cyclin D3 expression increased with 2004 WHO grading categories in stage Ta (p = 0.035, ANOVA) and stage T1 (p = 0.047, t test) tumours. Cyclin D3 was not related to other clinicopathological parameters, G1-S phase modulators, or 9p21 LOH. Cox's multivariate analysis selected cyclin D3 as an independent predictor of progression-free survival (p = 0.0012, relative risk (RR) = 5.2366) together with tumour size (p = 0.0115, RR = 4.4442) and cyclin D1 (p = 0.0065, RR = 3.3023). Cyclin D3 expression had the highest risk ratio. Our results suggest that expression of cyclin D3 is relevant to the progression-free survival of patients with Ta/T1 bladder carcinomas. Copyright (c) 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Squamous differentiation (SqD) is variably present in urinary tract tumours, but its significance remains unclear. In this study, SqD was assessed by immunohistochemistry using the monoclonal antibody Mac387 in 145 urothelial tumours (bladder, n = 115; renal pelvis, n = 30). Mac387 detects the myelomonocytic L1 antigen; a member of the calgranulin family shared by epithelial cells and keratinocytes. L1 antigen was shown in SqD in urothelial carcinomas of the bladder or the renal pelvis, including 11 cases with focal SqD unrecognised by conventional analysis. SqD is more frequent in renal pelvic tumours (p = 0.027) and increases with grade/stage mainly in bladder carcinoma (grade, p = 0.05; stage, p = 0.005). Stage Ta/T1 bladder carcinomas with SqD recurred more (p = 0.021). In conclusion, Mac387 efficiently shows SqD in urothelial tumours.

We examined the presence of human herpesvirus type 6 (HHV6) DNA in a series of 74 bladder carcinomas from a Mediterranean population to elucidate their possible role as cofactor in the development of bladder cancer with or without associated human papillomavirus (HPV) infection. HHV-6 type B DNA was present in 5 men (6.8%) out of the 74 tumors investigated; two of them had associated HPV-16 DNA in the same specimen. In one case that had associated urothelial carcinoma in situ, both HHV-6B and HPV-16 DNA were present. In conclusion, the low incidence of HHV-6B in bladder cancer and the ubiquitous nature of HHV-6 infection are more consistent with a bystander role rather than cofactor in the oncogenesis of bladder cancer.

We studied 159 cases of superficial (stage Ta or T1) bladder tumors to determine the significance on survival of a subset of regulators of transition from G1 to S phase of the cell cycle (p53, p21Waf1, p27Kip1, cyclin D1, cyclin D3) and tumor proliferation (Ki-67 [MIB-1]). Clinical findings (patient age, sex, tumor size, grade, stage [Ta or T1]) were included in the analysis. Univariate analysis revealed association of tumor size (P = .0353), grade in stage Ta tumors ( P = .0074), cyclin D1 expression (P = .0182), and Ki-67 index ( P = .0033) with disease-free survival and of tumor size ( P = .0005), stage (P = .0494), cyclin D3 expression ( P = .0105), and Ki-67 index ( P = .0272) with overall survival. Cox multivariate analysis revealed cyclin D1 expression and high proliferation index (disease-free) and tumor size, cyclin D3 expression, and high proliferation index (overall survival) as independent predictors. Results suggest that alterations of the progression from the G1 to S phase of the cell cycle are common in papillary urothelial bladder tumors. High tumor proliferation, expression of cyclins D1 and D3, and tumor size at diagnosis might be relevant predictors of survival in patients with stage Ta and T1 bladder urothelial tumors.

Prostaglandin E(1) (PGE(1)) reduces cell death in experimental and clinical manifestations of liver dysfunction. Nitric oxide (NO) has been shown to exert a protective or noxious effect in different experimental models of liver injury. The aim of the present study was to investigate the role of NO during PGE(1) protection against D-galactosamine (D-GalN) citotoxicity in cultured hepatocytes. PGE(1) was preadministered to D-GalN-treated hepatocytes. The role of NO in our system was assessed by iNOS inhibition and a NO donor. Different parameters related to apoptosis and necrosis, NO production such as nitrite+nitrate (NO(x)) release, iNOS expression, and NF-kappaB activation in hepatocytes were evaluated. The inhibition of iNOS reduced apoptosis induced by D-GalN in hepatocytes. PGE(1) protection against D-GalN injury was associated with its capacity to reduce iNOS expression and NO production induced by D-GalN. Nevertheless, iNOS inhibition showed that protection by PGE(1) was also mediated by NO. Low concentrations of a NO donor reduced D-GalN injury with a decrease in the extracellular NO(x) concentration. High concentrations of the NO donor enhanced NO(x) concentration and increased cell death by D-GalN. The present study suggests that low NO production induced by PGE(1) preadministration reduces D-GalN-induced cell death through its capacity to reduce iNOS expression and NO production caused by the hepatotoxin.

Objective: To determine the prognostic value of a subset of regulators of the transition from G1-to-S phase of cell cycle in stage T1 grade 3 bladder cancers.Methods: Fifty-one such cases were investigated to determine the significance on patient's survival of p53, p21 Waf1, p27Kip1, Cyclin D1, Cyclin D3, and ki67-MIB1 (proliferation index). The statistical analysis included Kaplan-Meier methodology with Log-rank test and Cox' proportional hazard analysis.Results: Tumor size (p = 0.0034), and the labeling index of ki67-MIb1 (p = 0.0034), p53 (p = 0.0332), p27kip1 (p = 0.0059) and Cyclin D1 (p = 0.0103) were associated to disease-free survival. Progression-free survival was related to tumor size (p < 0.0001), ki67 (p = 0.0163), p53 (p = 0.0041), p27kip1 (p = 0.0161), Cyclin D1 (p < 0.0001) and Cyclin D3 (p < 0.0001). Patient's overall survival was associated to Cyclin D3 (p < 0.0001), p53 (p = 0.0017), p21Waf1 (p = 0.0142), Cyclin D1 (p < 0.0001), ki67-MIB1 (p = 0.0450), and tumor size (p = 0.0296). Down-regulation of p27kip1 and Cyclin D3 over-expression (disease-free), over-expression of p53, Cyclin D1 and Cyclin D3 (progression-free), and over-expression of Cyclin D3 (overall survival) were independent predictors by Cox's multivariate analysis. Down-regulation of p27kip1 (p < 0.001, R.R. 0.997, 95%C.I. 0.995-0.999), and over-expression of Cyclin D1 (p < 0.001, R.R. 1.009, 95%C.I. 1.004-1.014) and Cyclin D3 (p = 0.005, R.R. 1.013, 95%C.I. 1.004-1.022) were the main independent predictors.Conclusion: Down-regulation of p27kip1 and over-expression of Cyclin D1 and Cyclin D3 might be relevant predictors of survival in stage T1 grade 3 bladder cancers, thus selecting a group of patients at higher risk of malignant behavior. (C) 2003 Elsevier B.V. All rights reserved.

d -galactosamine ( d -GalN) toxicity is a useful experimental model of liver failure in human. It has been previously observed that PGE 1 treatment reduced necrosis and apoptosis induced by d -GalN in rats. Primary cultured rat hepatocytes were used to evaluate if intracellular oxidative stress was involved during the induction of apoptosis and necrosis by d -GalN (0-40 mM). Also, the present study investigated if PGE 1 (1 w M) was equally potent reducing both types of cell death. The presence of hypodiploid cells, DNA fragmentation and caspase-3 activation were used as a marker of hepatocyte apoptosis. Necrosis was measured by lactate dehydrogenase (LDH) release. Oxidative stress was evaluated by the intracellular production of hydrogen peroxide (H 2 O 2 ), the disturbances on the mitochondrial transmembrane potential (MTP), thiobarbituric-reacting substances (TBARS) release and the GSH/GSSG ratio. Data showed that intermediate range of d -GalN concentrations (2.5-10 mM) induced apoptosis in association with a moderate oxidative stress. High d -GalN concentration (40 mM) induced a reduction of all parameters associated with apoptosis and enhanced all those related to necrosis and intracellular oxidative stress, including a reduction of GSH/GSSG ratio and MTP in comparison with d -GalN (2.5-10 mM)-treated cells. Although PGE 1 reduced apoptosis induced by d -GalN, it was not able to reduce the oxidative stress and cell necrosis induced by the hepatotoxin in spite to its ability to abolish the GSH depletion.

Lymphoepithelioma-like carcinoma (LELCA) of the urinary bladder is a rare variant of bladder cancer characterized by a malignant epithelial component densely infiltrated by lymphoid cells. It is characterized by indistinct cytoplasmic borders and a syncytial growth pattern. These neoplasms deserve recognition and attention, chiefly because they may be responsive to chemotherapy. We report on the clinicopathologic features of 13 cases of LELCA recorded since 1981. The chief complaint in all 13 patients was hematuria. Their ages ranged from 58 years to 82 years. All tumors were muscle invasive. A significant lymphocytic reaction was present in all of these tumors. There were three pure LELCA and six predominant LELCA with a concurrent transitional cell carcinoma (TCC). The remainder four cases had a focal LELCA component admixed with TCC. Immunohistochemistry showed LELCA to be reactive against epithelial membrane antigen and several cytokeratins (CKs; AE1/AE3, AE1, AE3, CK7, and CK8). CK20 and CD44v6 stained focally. The lymphocytic component was composed of a mixture of T and B cells intermingled with some dendritic cells and histiocytes. Latent membrane protein 1 (LMP1) immunostaining and in situ hybridization for Epstein-Barr virus were negative in all 13 cases. DNA ploidy of these tumors gave DNA histograms with diploid peaks (n=7) or non-diploid peaks (aneuploid or tetraploid; n=6). All patients with pure and 66% with predominant LELCA were alive, while all patients having focal LELCA died of disease. Our data suggest that pure and predominant LELCA of the bladder appear to be morphologically and clinically different from other bladder (undifferentiated and poorly differentiated conventional TCC) carcinomas and should be recognized as separate clinicopathological variants of TCC with heavy lymphocytic reaction relevant in patient management.

BACKGROUND:Tumour necrosis factor alpha (TNF-alpha) and nitric oxide modulate damage in several experimental models of liver injury. We have previously shown that protection against D-galactosamine (D-GalN) induced liver injury by prostaglandin E(1) (PGE(1)) was accompanied by an increase in TNF-alpha and nitrite/nitrate in serum.AIMS:The aim of the present study was to evaluate the role of TNF-alpha and nitric oxide during protection by PGE(1) of liver damage induced by D-GalN.METHODS:Liver injury was induced in male Wistar rats by intraperitoneal injection of 1 g/kg of D-GalN. PGE(1) was administered 30 minutes before D-GalN. Inducible nitric oxide synthase (iNOS) was inhibited by methylisothiourea (MT), and TNF-alpha concentration in serum was lowered by administration of anti-TNF-alpha antibodies. Liver injury was evaluated by alanine aminotransferase activity in serum, and histological examination and DNA fragmentation in liver. TNF-alpha and nitrite/nitrate concentrations were determined in serum. Expression of TNF-alpha and iNOS was also assessed in liver sections.RESULTS:PGE(1) decreased liver injury and increased TNF-alpha and nitrite/nitrate concentrations in serum of rats treated with D-GalN. PGE(1) protection was related to enhanced expression of TNF-alpha and iNOS in hepatocytes. Administration of anti-TNF-alpha antibodies or MT blocked the protection by PGE(1) of liver injury induced by D-GalN.CONCLUSIONS:This study suggests that prior administration of PGE(1) to D-GalN treated animals enhanced expression of TNF-alpha and iNOS in hepatocytes, and that this was causally related to protection by PGE(1) against D-GalN induced liver injury.