Activation of extracellular regulated kinases (ERK1/2) predicts poor prognosis in urothelial bladder carcinoma and is not associated with B-Raf gene mutations.

AIM: The analysis of the presence of B-Raf gene mutations in relation to ERK1/2 activation in bladder urothelial carcinoma (UC), in order to determine their potential role in tumour aggressiveness and patients' survival. METHODS: Polymerase chain reaction-single strand confirmation polymorphism (PCR-SSCP) and sequencing analysis were used for B-Raf gene mutation detection. pERK1/2 and FGFR3 expression were examined by immunohistochemistry in 152 and 116 primary UCs, respectively. RESULTS: None of the cases displayed mutations in exon 15 of B-Raf gene. Nuclear or cytoplasmic pERK immunoreactivity was displayed in 99.3% and 96.7% of cases, respectively. pERK nuclear expression increased with histological grade and with T-category. Nuclear and cytoplasmic pERK expression was unrelated to FGFR3 expression. In univariate survival analysis of muscle-invasive carcinomas, advanced T-category and higher pERK nuclear expression (p = 0.018) adversely affected survival. However, multivariate analysis in non-invasive as well as in muscle-invasive carcinomas selected only T-category as a significant prognosticator. CONCLUSIONS: Our findings suggest that elevated pERK expression occurs in UCs in the absence of B-Raf mutations and is not correlated with FGFR3 over-expression. Moreover, it implicates ERK activation in the acquisition of a more aggressive phenotype. However, the assessment of pERK1/2 expression does not seem to add to the prognostic information provided by classical prognosticators.

Expression of nuclear factor-kappaB in human astrocytomas: relation to pI kappa Ba, vascular endothelial growth factor, Cox-2, microvascular characteristics, and survival.

Although NF-kappaB has been reported to be constitutively activated in various neoplasms, little information is available about its clinical significance in astrocytomas. In this study, we investigated the association of NF kappa B1/p50 and pI kappa Ba immunohistochemical expression with clinicopathologic features, vascular endothelial growth factor, Cox-2, and microvascular parameters in paraffin-embedded tissue from 82 patients with astrocytomas. pI kappa Ba expression was positively correlated with nuclear (P = .0010) and negatively with cytoplasmic (P = .0008) NF kappa B1/p50 expression. Nuclear NF kappa B1/p50 and pI kappa Ba expression increased with tumor grade (P = .0001 and P < .0001). Nuclear NF kappa B1/p50 was associated with vascular endothelial growth factor (P = .0079), Cox-2 (P = .0500), and total vascular surface area (P = .0430), although the latter was significant only in grades II and III. pI kappa Ba was also positively correlated with microvessel caliber (pI kappa Ba/area; P = .0087). Multivariate analysis selected NF kappa B/p50 expression as an independent prognosticator not only for the entire cohort (P = .0220), but also for grades II and III (P = .0029) and grade IV cases (P = .0310). Our results suggest that nuclear NF kappa B1/p50 expression is dictated by its interaction with I kappa Ba in astrocytomas and is associated with tumor grade and angiogenic factors, denoting the importance of nuclear NF kappa B/p50 expression in patients' prognosis.

Gastrointestinal stromal tumor of the gastric serosa protruding to the greater omentum. Report of a giant tumor with a rare histological pattern.

We report an unusual case of a 76 year old woman admitted to our hospital for investigation of anemia and palpable epigastric mass. Intraoperatively a huge (19 cm), well defined tumor was found adhering to the stomach wall, protruding into the greater omentum and compressing the transverse colon. A tumorectomy was performed and the greater omentum was removed due to its close relation. Pathology revealed a high risk Gastrointestinal Stromal tumor of the gastric serosa. Histologically the tumor was of mixed type (spindle and epithelioid cells) with hem-angio-peri-cytomatoid pattern peripherally, variably myxoid stroma, central necrosis and cytologic pleomorphism. On immunohistochemical examination there was a consistent positivity for c-kit (CD-117) and CD-34, but without myogenic or neural differentiation. We consider this case unusual because of its huge size, its gastric serosal location and its extremely rare histological pattern.

TGF-betaRII, BAX, IGFIIR, caspase-5, hMSH3 and hMSH6 alterations are not associated with microsatellite instability or p53 mutations in invasive urothelial carcinoma of the urinary bladder.

AIM: The aim of this study was to determine the potential synchronous contribution of alterations in TGF-betaRII, BAX, IGFIIR, caspase-5, hMSH3 and hMSH6 genes to the development and clinical outcome of bladder cancer, in relation to p53 mutations, microsatellite status and hMLH1/hMSH2 expression. METHODS: Molecular biology techniques as well as immunohistochemistry were applied in 69 samples from patients with urothelial carcinoma. RESULTS: Microsatellite alterations were observed in TGF-betaRII(A)(10 )(16%) and BAX(G)(8 )(3%), irrespective of the presence of p53 mutations, but not in IGFIIR(G)(8), caspase-5(A)(10, ) hMSH3(A)(8) and hMSH6(C)(8). A statistically significant correlation could be found only between hMLH1 expression and the presence of microsatellite instability (Fisher's exact test, p = 0,013). Survival analysis indicated that apart from grade and T-category, hMLH1 expression was the only parameter significantly affecting overall survival (p = 0.021 in univariate and p = 0.015 in multivariate analysis) and recurrence-free survival (p = 0.0463 in univariate and p = 0.022 in multivariate analysis). CONCLUSIONS: We conclude that alterations of the examined target genes of MSI are rare in urinary bladder carcinoma and they are not associated with microsatellite instability or the presence of p53 mutations.

Expression of hypoxia-related tissue factors in astrocytic gliomas. A multivariate survival study with emphasis upon carbonic anhydrase IX.

Carbonic anhydrase IX (CAIX) is a transmembrane enzyme involved in the reversible metabolism of carbon dioxide to carbonic acid and, hence, in physiological pH regulation. It also participates in cellular differentiation and proliferation, its expression being absent in most normal tissues. It has been recently postulated that the hypoxia-inducible factor (HIF-1) pathway up-regulated by hypoxia accounts for CAIX overexpression in most human tumors. In the present study, we examined the expression of this enzyme in diffuse gliomas of astrocytic origin in relation to vascular endothelial growth factor (VEGF) and HIF-1alpha expression, proliferation rate (as assessed with Ki-67 antigen), microvessel morphology, and survival. Of 84 cases analyzed, 61 cases (72.6%) displayed strong membrane and/or cytoplasmic expression of CAIX and were grouped as positive. Immunoreactivity tended to have a perinecrotic distribution and increased in parallel with the extent of necrosis (P < .001) and histologic grade (P < .001). A positive correlation was also noted with HIF-1alpha and VEGF expression (P < .001), proliferation rate (P = .010), microvessel density (P = .004), and microvessel caliber parameters (P = .014-.038). In univariate survival analysis, increased CAIX expression was associated with shortened survival in the entire cohort (P < .0001), along with VEGF (P = .0205) and HIF-1alpha levels (P = .0190). Multivariate analysis selected the interaction model of CAIX, with grade and age as the only parameters independently affecting survival. CAIX expression was also the only significant parameter for the survival of patients with grades II/III. We conclude that CAIX may be used as a prognostic indicator in diffuse astrocytomas to refine the information provided by grade. Given the role of CAIX in the acidification of tumor environment and its up-regulation by hypoxia, it is thought that CAIX expression may be linked to resistance of tumor cells to radiotherapy by allowing them to acclimatize to a hypoxic and acidic microenvironment.

Cyclooxygenase-2 expression in astrocytomas. Relationship with microvascular parameters, angiogenic factors expression and survival.

OBJECTIVE: Cyclooxygenase-2 (COX-2) is the enzyme isoform involved in the synthesis of prostaglandins (PGs) and thromboxane from arachidonic acid. The role of the up-regulation of COX-2 in the formation and progression of gliomas has been dealt with in earlier reports, which describe increased levels of PGs within gliomas. In the present study, we examined the expression of COX-2 in diffuse gliomas of astrocytic origin in relation to microvascular parameters, angiogenic factors and survival. MATERIALS AND METHODS: A total of 83 cases of diffuse astrocytomas (grade II-IV) were analyzed by immunohistochemistry for the presence of COX-2. RESULTS: COX-2 expression was detected in 79 cases (95%) with an increased expression in grade IV as compared to grades II/III (p=0.024). A positive correlation occurred between COX-2 and angiogenic factors such as vascular endothelial growth factor (VEGF) (p<0.0001) and hypoxia inducible factor (HIF)-1alpha (p=0.005), as well as the tumours' proliferative activity (expressed as the percentage of Ki-67 positive cells) (p=0.032), and total vascular area (TVA) (p=0.040). In univariate analysis, COX-2 was associated with shortened survival (p = 0.050). Multivariate survival analysis showed that the interaction model of COX-2 with grade along with age were the only significant prognostic indicators. CONCLUSION: These results implicate COX-2 in the angiogenesis and biological aggressiveness of diffuse astrocytomas, and suggest that it would be worthwhile to examine how the inhibition of COX-2 expression may influence astrocytoma patients' survival.

Minichromosome maintenance proteins 2 and 5 expression in muscle-invasive urothelial cancer: a multivariate survival study including proliferation markers and cell cycle regulators.

Evaluation of cell cycle regulators has gained special interest in the effort to increase the amount of prognostic information in malignant tumors. Minichromosome maintenance proteins (MCMs) drive the formation of prereplicative complexes, which is the first key event during G1 phase. Therefore, altered MCM expression may be a hallmark of cell cycle deregulation, which is supposed to be the most essential mechanism in the development and progression of bladder cancer. Our aim was to investigate the value of MCMs as proliferation markers and prognostic indicators in detrusor muscle-invasive urothelial bladder carcinomas. We analyzed immunohistochemically the expression of MCM-2 and MCM-5 in 65 patients with detrusor muscle-invasive urothelial bladder carcinomas in relation with clinicopathologic parameters, patients' overall and disease-free survival, and the expression of the conventional proliferation index Ki-67 and other cell cycle modulators (p53, pRb, p21(WAF1), and p27(Kip1)). The levels of MCM-2 and MCM-5 were significantly higher in high-grade (P < .0001), advanced-stage (P = .001), and nonpapillary tumors (P < .0001). The expression of MCM-2 and MCM-5 significantly associated with the conventional proliferation index Ki-67 (P = .0001 for each protein). The expression of MCM-2 or MCM-5 positively correlated with p53 labeling index (P = .014 and P = .009, respectively). Also, median p21(WAF1) labeling index was higher in MCM-5 high expressors (P = .028). Finally, both MCM-2 and MCM-5 associated significantly with adverse patients' outcome in both univariate (P = .0072 and P = .0074, respectively) and multivariate (P = .0001) analysis. In conclusion, MCM-2 and MCM-5 appear to be reliable proliferation indexes and useful prognostic markers in patients with muscle-invasive urothelial bladder carcinomas.

c-FLIP expression in bladder urothelial carcinomas: its role in resistance to Fas-mediated apoptosis and clinicopathologic correlations.

OBJECTIVES: To investigate the incidence of Fas (exon 9) mutations and the expression of Fas, Fas-Fas ligand (FasL) system, and cellular FLICE-like inhibitory protein (c-FLIP) in relation to standard clinicopathologic parameters and patient outcome in bladder carcinoma. Disruption of apoptotic cell death has been implicated in tumor aggressiveness in bladder urothelial carcinomas. The FasL system is involved in the execution of apoptosis induced by the immune system. c-FLIP protein constitutes an important endogenous inhibitor of Fas and other death receptor-mediated apoptosis. METHODS: The expression of Fas, FasL, and c-FLIP was quantified immunohistochemically in paraffin-embedded tissues from 53 patients for whom clinical information was available. DNA extracted from the same samples was screened for mutations in Fas exon 9 by single-strand conformation polymorphism and sequencing. The effect of Fas, FasL, and c-FLIP on clinical outcome was assessed by univariate and multivariate analyses. RESULTS: Positive immunostaining was detected for Fas, FasL, and c-FLIP in 72%, 66%, and 81% of cases, respectively. Concurrent expression of Fas and FasL was seen in 27 samples (51%), of which 22 (81.5%) also displayed c-FLIP positivity. FasL and c-FLIP expression increased with advancing stage but was absent from normal urothelium. None of the 53 urothelial carcinoma samples analyzed showed evidence of mutations by polymerase chain reaction single-strand conformation polymorphism and direct sequencing. Survival analysis demonstrated that although both FasL and c-FLIP expression adversely affected survival, only c-FLIP remained statistically significant on multivariate analysis. CONCLUSIONS: The frequent expression and coexpression of Fas, FasL, and c-FLIP in urothelial carcinomas implicates c-FLIP as an inhibitor of the Fas-FasL-induced death pathway in these tumors. Moreover, c-FLIP conveys independent prognostic information in the presence of classical prognosticators.

Differential expression of bcl-2 family proteins in bladder carcinomas. Relationship with apoptotic rate and survival.

OBJECTIVE: To elucidate the role of various bcl-2 family molecules in the regulation of apoptosis and the progression of urothelial cancer, in relation to standard prognosticators. METHODS: Paraffin-embedded archival tissue from 103 N0M0 consecutive patients with invasive bladder cancer (28 T1, 57 T2, 13 T3 and 5 T4) was immunostained for bcl-2, bax, bcl-XL, bcl-Xs, p53, Ki-67 and with an anti-single stranded DNA monoclonal antibody recognizing the apoptotic cells. Survival analysis was restricted to T2-T4 tumours. Patients were followed-up until death (n = 27) or for a mean (+/- S.D.) follow-up of 37.6 (+/- 17.4) months. Within this period, 39 patients relapsed after a mean (+/- S.D.) period of 13.6 (+/- 12.3) months. RESULTS: Most tumours were immunoreactive for bax (73.1%) and bcl-XL (80.9%) whereas bcl-2 and bcl-XS expression was comparatively less common (44.4 and 28.9%, respectively). The bcl-XL and bcl-XS positivity was related to high grade (P = 0.007) and advanced stage (P = 0.010), respectively. On the contrary, bax and bcl-2 positivity was unrelated to stage or grade. Apoptotic rate was independently influenced only by p53, bcl-2 and proliferation rate. In multivariate analysis of T2-T4 urothelial carcinomas (UC)s, only bax along with T-category and age were the significant predictors of disease-free survival. Increased apoptosis and T-category were also independently related to the overall survival in T2-T4 UCs. CONCLUSIONS: The expression of bcl-2 family members appears to be differentially regulated in association with UC evolution. Most importantly, bax immunostaining offers additional information to that provided by traditional prognosticators, with regard to disease-free survival of T2-T4 UCs.