TRAIL and osteoprotegerin (OPG) expression in bladder urothelial carcinoma: correlation with clinicopathological parameters and prognosis.

AIMS: To investigate the expression of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and osteoprotegerin (OPG) in bladder urothelial carcinomas (UCs) and assess possible interrelations with other regulators of TRAIL induced apoptosis (p65/NF-κB, p-ERK1/2, p-AKT) and FGFR3, as well as to elucidate their potential involvement in bladder tumourigenesis and determine their potential prognostic utility. METHODS: Paraffin embedded transurethral resection tissue from 128 patients with UC was immunostained for TRAIL and OPG as well as for p65/NF-κB, p-ERK1/2, p-AKT and FGFR3. RESULTS: TRAIL and OPG were coexpressed in 96.6% of cases and positively interrelated. OPG expression was significantly different among histological grades, being higher in low-grade UCs and was inversely correlated with the presence of lymphovascular invasion (LVI). TRAIL also displayed an inverse relationship with histological grade, T-category and LVI. Both OPG and TRAIL expression were positively correlated with FGFR3 expression, the former relationship being marginal. Moreover, increased TRAIL expression was marginally correlated with lower NF-κB/p65 nuclear expression. Increased OPG expression adversely affected survival both in univariate and multivariate analysis. CONCLUSIONS: OPG and TRAIL are frequently expressed and coexpressed in UCs, supporting the involvement of OPG in the resistance to TRAIL-driven apoptosis. Inhibition of NF-κB activation may also play a similar role, although less important. OPG emerged as an independent prognostic marker of adverse significance.

A comprehensive immunohistochemical and molecular approach to the PI3K/AKT/mTOR (phosphoinositide 3-kinase/v-akt murine thymoma viral oncogene/mammalian target of rapamycin) pathway in bladder urothelial carcinoma.

UNLABELLED: What's known on the subject? and What does the study add? A few published studies investigating single or various PI3K/AKT/mTOR signalling components have produced inconsistent results. Moreover, PI3K regulatory subunit p85a and activated p70S6K expression levels have not been previously examined in urothelial carcinoma (UC). The present study addresses simultaneously all key members of PI3K/AKT/mTOR signalling cascade supporting a differential implication of PI3K/AKT/mTOR pathway components in urothelial tumorigenesis. Furthermore, we propose p-4E-BP1 as a potential prognostic marker in UC, which might assist the selection of patients more likely to benefit from chemotherapy regimens based on PI3K/AKT/mTOR pathway inhibition. Finally, the present study indicates PIK3CA/AKT1 mutational status as a potential predictive marker for time-to-recurrence. OBJECTIVE: • To perform a comprehensive simultaneous assessment of all key members of phosphoinositide 3-kinase/v-akt murine thymoma viral oncogene/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway along with AKT homolog 1 (AKT1) and PIK3 catalytic alpha polypeptide (PIK3CA) mutations in bladder urothelial carcinoma (UC). • Published information is limited to a few studies looking into single or various combinations of members of this pathway with inconsistent results. In particular the expression status of phosphorylated (p-)p70S6 kinase (p70S6K) and p85a subunit of PI3K has not been tested in UC. PATIENTS AND METHODS: • Paraffin-embedded transurethral resection tissue from 113 patients with UC was investigated for the association of p85aPI3K, p-AKT, p-mTOR, p-p70S6K and p-4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) expression status, as well as PIK3CA and AKT1 mutations with p-extracellular signal-regulated kinase 1/2 (ERK1/2), fibroblast growth factor receptor 3 (FGFR3), pathological features, recurrence and cancer-specific survival. RESULTS: • With the exception of p-p70S6K, all others components of the PI3K/AKT/mTOR pathway were upregulated in UCs as compared with normal urothelium. • p-mTOR expression strongly correlated with its upstream p-AKT and marginally with its downstream p-p70S6K. p85aPI3K and p-ERK1/2 levels were also marginally correlated. • PIK3CA and AKT1 mutations were distinctly uncommon and mutually exclusive, without any association with pathological features. However, the presence of AKT1 mutations was associated with increased FGFR3 levels and was restricted to p85aPI3K immunonegative cases, whereas PIK3CA mutant cases had marginally lower p85aPI3K levels. • The presence of PIK3CA single or combined with AKT1 mutations was associated with shorter recurrence-free survival in univariate survival analysis. An inverse relationship was established between p-4E-BP1 immunopositivity and histological grade or T category, as well as between p-p70S6K levels and T category, the latter relationship being of marginal significance. • p-4E-BP1 nuclear expression was marginally associated with the presence of lymphovascular invasion and adversely affected survival in multivariate, but not in univariate analysis. CONCLUSIONS: • PI3K/AKT/mTOR signalling components appear to be differentially implicated in urothelial tumorigenesis and, with the exception of p85aPI3K, are unrelated to the PIK3CA or AKT1 mutational status. • Our findings propose p-4E-BP1 as a potential prognostic marker in UC independent of its association with pathological features, which might assist the selection of patients more likely to benefit from PI3K/AKT/mTOR axis inhibition. • PIK3CA/AKT1 mutational status may have a place in the prediction of time-to-recurrence.

Prognostic significance of replication protein A (RPA) expression levels in bladder urothelial carcinoma.

OBJECTIVE: To elucidate the role of replication protein A (RPA) in both superficial (Ta-T1) and muscle-invasive (T2-T4) urothelial carcinomas (UCs), investigating its potential prognostic usefulness. PATIENTS AND METHODS: Paraffin-embedded tissue from 156 patients with bladder UC was immunostained for RPA1 and RPA2. RESULTS: RPA1 and RPA2 labelling indexes (LIs) decreased with increasing histological grade (both P < 0.001) and T-category in the entire cohort (P = 0.008 and P < 0.001, respectively) and in muscle-invasive carcinomas (P = 0.014 and P = 0.012, respectively). RPA1 expression was positively correlated with RPA2 (Spearman's correlation coefficient ρ = 0.309, P < 0.001). Both RPA1 and RPA2 LIs were positively correlated with cyclin D1 expression (ρ = 0.354, P < 0.001 and ρ = 0.934, P < 0.001). In survival analysis of the entire cohort decreased RPA2 and RPA1 correlated with a lesser probability of survival (P < 0.001 and P = 0.018). In non-muscle-invasive tumours (Ta-T1) only lower RPA2 (P < 0.001) was correlated with shortened survival, whereas in muscle-invasive tumours (T2-T4) decreased RPA2 and RPA1 expression levels were associated with adverse prognosis (P = 0.035 and P = 0.042, respectively). In multivariate survival analysis of the entire cohort and in non-muscle-invasive cases RPA2 expression remained significant, even when adjustment for cyclin D1 expression was applied. CONCLUSIONS: RPA1 and RPA2 overexpression seems to be more important during early T-categories of bladder carcinogenesis. Showing similar kinetics with cyclin D1. RPA2 expression emerges as a valuable marker of favourable prognosis in the entire cohort and in non-muscle-invasive tumours, supplementing the information obtained by standard clinicopathological prognosticators.

D-type cyclins in superficial and muscle-invasive bladder urothelial carcinoma: correlation with clinicopathological data and prognostic significance.

PURPOSE: To elucidate the role of D-type cyclins in both superficial (Ta-T1) and muscle-invasive (T2-T4) urothelial carcinomas (UCs), investigating their potential prognostic usefulness. METHODS: Paraffin-embedded tissues from 157 patients with bladder UC were immunostained for cyclins D1, D2 and D3. RESULTS: Cyclin D1 expression positively correlated with D2 and negatively with D3. Cyclin D1 expression decreased with increasing grade (P = 0.0001) and tumour T-category in the entire cohort and in muscle-invasive carcinomas (P = 0.0001 and P = 0.0033). Cyclin D2 correlated with grade (P = 0.0005) and T-category (P = 0.0078), a relationship which remained significant in muscle-invasive (P = 0.0135). Cyclin D3 immunoreactivity increased with histologic grade and T-category in the entire cohort (P = 0.0001 in both relationships), in superficial (P = 0.0034) and in muscle-invasive carcinomas (P = 0.0036, respectively). Survival analysis in superficial tumours showed that higher cyclin D1 (P = 0.0001) and higher cyclin D3 levels (P = 0.0032) were correlated with a lesser probability of survival. In muscle-invasive tumours, lower cyclin D1 (P = 0.0234), and D2 (P = 0.0424) and higher cyclin D3 (P = 0.0322) correlated with shortened survival. In multivariate analysis in superficial tumours only cyclin D3 expression remained significant. Cyclin D3 expression also retained its adverse significance in muscle-invasive tumours. CONCLUSIONS: Cyclin D1 overexpression seems to be more important during early T-categories of bladder carcinogenesis, whereas cyclin D3 is implicated in the acquisition of a more aggressive phenotype. Cyclin D3 overexpression emerges as an independent adverse prognostic marker in both superficial and muscle-invasive tumours. Cyclin D1 is an independent indicator of shortened survival only in muscle-invasive tumours.

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.

Vascular fractal dimension and total vascular area in the study of oral cancer.

BACKGROUND: Microvessel quantification has been studied extensively as a factor reflecting angiogenesis in various malignant tumors. The aim of our study was to evaluate the vascular fractal dimension and the immunohistochemically positive total vascular area in oral cavity carcinomas in order to assess their potential value as factors reflecting angiogenesis. METHODS: Histologic sections from 48 carcinomas and 17 nonmalignant mucosa specimens were evaluated by image analysis using fractal analysis software. Total vascular area was also quantified. RESULTS: Carcinomas presented higher mean values of vascular fractal dimension and total vascular area compared to normal mucosa. The difference for the vascular fractal dimension was statistically significant. CONCLUSIONS: This study provides evidence that vascular fractal dimension could be used as a reliable factor reflecting angiogenesis in oral squamous cell carcinoma and that there are several statistically significant correlations among total vascular area, vascular fractal dimension, nuclear size, and clinicopathologic factors.

Clinical significance of nuclear factor (NF)-kappaB levels in urothelial carcinoma of the urinary bladder.

Nuclear factor (NF)-kappaB has been reported to be constitutively activated in various human neoplasms. However, its clinical significance in bladder urothelial carcinoma (UC) remains an unresolved issue. We conducted this study trying to elucidate the role of NFkappaB in bladder UC and its potential prognostic significance, by quantifying immunohistochemically the levels of p65/RelA expression in paraffin-embedded tissue from 116 patients. Some of the cases had previously been stained for cellular FLICE-like inhibitory protein (c-FLIP) and bcl-2. Seventy-four cases displayed concurrent cytoplasmic and nuclear immunoreactivity, whereas 18 only nuclear immunoexpression and 21 only cytoplasmic immunoexpression, and the remaining three cases were negative for p65/RelA. Nuclear p65/RelA expression was positively associated with tumour grade and T-category (p=0.0001 in both cases). In addition, cytoplasmic p65/RelA expression was lower in advanced T-category (p=0.0030). Moreover, p65/RelA nuclear expression was positively correlated with c-FLIP (p=0.0109) and bcl-2 (p=0.0452). p65/RelA nuclear expression adversely affected survival in both univariate and multivariate analysis in superficial (Ta-T1; p=0.0010 and p=0.0008) as well as in muscle-invasive carcinomas (T2-T4; p=0.0004 and p=0.0003). Our results demonstrate that NF-kappaB nuclear expression is correlated with histologic grade and T category in bladder UC. Moreover, NF-kappaB nuclear expression emerges as an independent prognosticator of adverse significance, conveying information beyond that obtained by standard clinicopathological prognosticators.

Segmentation of transitional cell carcinoma nuclei by nonsupervised thresholding in different color spaces.

OBJECTIVE: To develop a method for nonsupervised thresholding of transitional cell carcinoma nuclei of hematoxylin-eosin-stained histologic sections. STUDY DESIGN: For grayscale, RGB, HSL and L*a*b* thresholding we used an extension of a clustering method, based on a between-class/within-class criterion, applying optimal gray-level thresholding to distributions of R, G, B, or H and S or L* color domains. Algorithms were tested on 20 hematoxylin-eosin-stained sections of bladder carcinomas. RESULTS: Results were compared with corresponding results of manually selected nuclear areas. Images were compared pixel to pixel with matching reference images. Grayscale automatic thresholding presented unacceptably low pixel specificity, which complicated further nuclear segmentation. Nonsupervised thresholding in RGB or HSL, as well as semimanual thresholding in L*a*b* color space demonstrated significantly better accuracy and high values of pixel specificity and sensitivity, which permitted errors of only 4.27-5.83% in the subsequent mean area estimation of the transitional cell carcinoma nuclei. CONCLUSION: Nonsupervised multispectral thresholding in RGB or HSL color space extends single graylevel thresholding techniques to multilevel thresholding. This seems to be an effective, relatively simple and fast alternative to the widely used automatic grayscale or manual color thresholding for segmentation of nuclei in routine histologic sections.

Malignant peripheral nerve sheath tumor of the urinary bladder in von Recklinghausen disease.

A case of malignant peripheral nerve sheath tumor of the bladder in a 57-year-old man with multiple neurofibromatosis type 1 is presented. The patient had a recent history of a transurethrally resected bladder ganglioneuroma. A probable histogenetic association between these two extremely rare neoplasms is proposed.