Expression of circadian clock genes and proteins in urothelial cancer is related to cancer-associated genes.

BACKGROUND: The purpose of this study was to evaluate invasive and metastatic potential of urothelial cancer by investigating differential expression of various clock genes/proteins participating in the 24 h circadian rhythms and to compare these gene expressions with transcription of other cancer-associated genes. METHODS: Twenty seven paired samples of tumour and benign tissue collected from patients who underwent cystectomy were analysed and compared to 15 samples of normal bladder tissue taken from patients who underwent cystoscopy for benign prostate hyperplasia (unrelated donors). Immunohistochemical analyses were made for clock and clock-related proteins. In addition, the gene-expression levels of 22 genes (clock genes, casein kinases, oncogenes, tumour suppressor genes and cytokeratins) were analysed by real-time quantitative PCR (qPCR). RESULTS: Considerable up- or down-regulation and altered cellular distribution of different clock proteins, a reduction of casein kinase1A1 (CSNK1A1) and increase of casein kinase alpha 1 E (CSNK1E) were found. The pattern was significantly correlated with simultaneous up-regulation of stimulatory tumour markers, and a down-regulation of several suppressor genes. The pattern was mainly seen in aneuploid high-grade cancers. Considerable alterations were also found in the neighbouring bladder mucosa. CONCLUSIONS: The close correlation between altered expression of various clock genes and common tumour markers in urothelial cancer indicates that disturbed function in the cellular clock work may be an important additional mechanism contributing to cancer progression and malignant behaviour.

Urokinase-type plasminogen activator receptor (uPAR) expression is associated with T-stage and survival in urothelial carcinoma of the bladder.

OBJECTIVES: To evaluate the expression-and localization pattern of the urokinase-type plasminogen activator receptor (uPAR), focusing on its clinical implications in patients with urothelial neoplasia of the bladder treated with radical cystectomy. uPAR is a central molecule in tissue remodeling during cancer invasion and metastasis and is an established prognostic marker in cancer. The expression and localization of uPAR and its prognostic significance is only limitedly investigated in urothelial bladder neoplasia. MATERIALS AND METHODS: The expression-and localization pattern of uPAR was investigated in formalin-fixed paraffin-embedded tumor tissue from 149 patients treated with radical cystectomy between 1988 and 2005. uPAR expression was determined by immunohistochemistry and scored as either negative or positive. Separate values were obtained for cancer cells, macrophages, and myofibroblasts at the invasive front and tumor core, respectively. Statistical analyses were performed to evaluate the association of uPAR localization and score with clinicopathologic covariates and survival. RESULTS: uPAR positivity was seen in 122/137 (89%) and 118/149 (74%) of the neoplasias at the invasive front and tumor core, respectively. uPAR was primarily expressed by myofibroblasts and macrophages in the surrounding stroma as well as some cancer cells. A significant association between uPAR positivity and T-stage as well as grade was found for all 3 cell types in tumor core (P ≤ 0.04 for all comparisons). In univariate analysis, the uPAR positive group had a shorter survival than the uPAR negative group (hazard ratio = 2.39; 95% CI: 1.15-5.01; P = 0.020). CONCLUSIONS: The expression of uPAR is a possible prognostic marker that could be useful in identification of patients with aggressive, highly invasive tumors that could benefit from additional chemotherapy or more intensive follow-up after cystectomy.

Time-dependent biological differences in molecular markers of high-grade urothelial cancer over 7 decades (ras proteins, pTEN, uPAR, PAI-1 and MMP-9).

The aim of this study was to evaluate changes and correlations between various molecular markers related to growth regulation and invasiveness in urothelial carcinomas in samples collected from 1932 to 2004. Paraffin-embedded autopsy/biopsy tissues from 144 patients were stained with antibodies against H-K-N ras proteins, pTEN protein, urokinase plasminogen activator receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase-9 (MMP-9) and analyzed by in situ hybridization. Statistical analysis was performed by SPSS using cross tabulation and logistic regression. While the presence of K-ras, N-ras, PAI-1, and loss of pTEN increased over the last few decades, uPAR expression decreased during the same period. The increase in K-ras expression associated positively with the increase in expression of the other two ras proteins, H-ras and N-ras, and the loss of pTEN. A strong positive correlation was also observed between PAI-1 and uPAR, PAI-1 and previously detected markers, EGFR (epidermal growth factor receptor) and p53. Presence of uPAR was found to be positively associated with p16 expression. Multivariate analysis with clinical parameters revealed a positive correlation between PAI-1 expression and tumour grade, CkHMW (high molecular weight cytokeratin) and tumour grade, CkHMW and metastasis, EGFR and metastasis. mRNA could be detected in samples from the last 50 years while older samples were negative, indicating its complete degradation during longer storage. In conclusion, increased accumulation of K-ras, N-ras, and PAI-1 together with loss of pTEN in bladder carcinomas of grades II and III seems to be more dominant in recent times, suggesting an altered malignant potential in these neoplasms.

Non-adhesive organ culture of human biliary epithelium with stroma.

OBJECTIVE: Explanted tissue has been shown to keep adult human cells in organ culture with a preserved morphology for at least one month as spheres in a non-adhesive organ culture. In the present study, we explored whether also human biliary epithelium can be grown in this manner, because the result may be of interest in studies of hepato-biliary-pancreatic carcinogenesis. MATERIAL AND METHODS. Small tissue samples were obtained from the gallbladder wall of patients who had been operated upon with cholecystectomy. Fragments of about 300 microm in diameter from each patient were cultured and investigated with light microscopy at the time of explantation and after 5, 10, 20, 30 and 40 days of culture. Scanning and transmission electron microscopy were performed to demonstrate the ultrastructure. Incubation of cultured fragments with the vital dyes revealed a viable epithelium. RESULTS: At the time of explantation, all the tissue fragments had a rough appearance with an uneven, torn periphery, while during the first few days of culture they became rounder with a smooth-looking surface covering the entire circumference. This spheroid morphology persisted for the remainder of the culture period. The core of the fragments harboured connective tissue with vascular elements, fibroblasts and leucocytes. Immunostaining for cytokeratin 7, 19 and 20 revealed a strong positive staining of the epithelium. CONCLUSIONS: These results show that biliary epithelium can be grown in vitro in a non-adhesive organ culture with their stroma.

Immunohistochemical markers in urinary bladder carcinomas from paraffin-embedded archival tissue after storage for 5-70 years.

OBJECTIVE: To evaluate archival tissue specimens from bladder tumours and seek molecular changes in samples collected over seven decades previously, as although the frequencies of some cancer types have remained stable during the last 50 years, the incidence of others, including bladder tumours, has increased significantly, and molecular analyses of bladder cancer over periods with an increasing incidence are of interest as the findings might reflect varying external influences. MATERIALS AND METHODS: Immunohistochemical staining with the biological markers p53 protein, p16 protein, epidermal growth factor receptor (EGFR), cytokeratin 7 and high molecular weight 34betaE12 cytokeratin (HMW-cytokeratin, characteristic of basal cells) was used on archival, paraffin wax-embedded autopsy/biopsy tissue material collected from 144 patients with invasive bladder cancer (World Health Organisation grade II and III). The cases were selected from the periods 1932-48, 1950-59, 1960-70 and 1990-2004. Control immunohistochemistry was done on available normal tissue (i.e. connective and fatty tissue, heart, lungs and normal urinary bladder epithelium) obtained from the autopsies. RESULTS: The normal tissues were all largely negative for EGFR, had <1% positively stained nuclei for p53 and strong positive reactions for p16, and in epithelial tissues the two cytokeratins were detected. The positive scores for HMW-cytokeratin in the tumour tissue decreased significantly from approximately 90% to 30% over the 70 years. For p53 there was a higher fraction of positive scores (borderline significant) with time. The p16-positive tumours showed no significant variation, with the highest frequency of positive scores in recent years. Overexpression of EGFR in the tumours was significantly correlated with the occurrence of HMW-cytokeratin and decreased from approximately 85% to 65% (not significant), with the lowest frequency in the samples from 1990 to 2004. Autolysis after death or long storage periods did not compromise good quality in the histochemical analyses of the autopsy tissue. CONCLUSION: The higher frequency of HMW-cytokeratin, lower p53 accumulation and more EGFR expression in grade II and III urinary bladder carcinomas from the 1930s could indicate different phenotypes in bladder cancer during this 70-year period. The successful detection of these protein markers in old archival material allows larger retrospective studies that might increase the understanding of molecular carcinogenesis in bladder cancer.

A concomitant tumour boost in bladder irradiation: patient suitability and the potential of intensity-modulated radiotherapy.

BACKGROUND AND PURPOSE: In radiotherapy (RT) of bladder cancer, dose escalation without increased adverse effects could be achieved with a concomitant bladder tumour boost. In this study we quantified (1) the fraction of patients suitable for this approach, and (2) the potential of intensity-modulated RT (IMRT) to achieve this boost while also sparing normal tissues. MATERIALS AND METHODS: The fraction of patients suitable for this boost approach was quantified using both a series of 30 radical therapy candidates, and a series of 15 consecutive RT patients. IMRT plans with 3, 5, 7 and 9 equi-spaced beams were set up for the patients in the RT series found suitable for a boost. Two sets of targets were defined, with (i) wide and (ii) narrow margins around both the tumour (prescribed 120% dose) and the non-involved bladder (prescribed 100% dose). The inverse planning optimisation minimised the dose deviation across the targets whilst fulfilling dose-volume histogram (DVH) constraints--based on what could be achieved with conformal RT (CRT)--for both the normal tissues and the targets. RESULTS: Fourteen of the 30 radical therapy candidates (47%) and 10 of the 15 RT patients (67%) were suitable for a boost. The 20% boost could be obtained while maintaining target coverage with at least one IMRT plan in 9 of 10 cases with wide margins and for all 10 cases with narrow margins. Using wide margins, all 3-field plans were unacceptable, the 5-field plans were acceptable for 5 of 10, and the 7- and 9-field plans for 9 of the 10 patients. The normal tissue volumes receiving doses >100% were on average reduced by a factor of 3-4 compared with CRT. The normal tissue volumes receiving intermediate doses (73-88%) decreased slightly, whereas volumes receiving the lowest doses (30-48%) increased with the number of beams. The use of narrow margins resulted in markedly lower normal tissue irradiation. CONCLUSION: This study has shown bladder tumour boosting to be both clinically relevant and technically feasible using IMRT. This approach is ready for clinical implementation, although further improvement could be expected if integrated with target localisation techniques.

Is preoperative serum prostate-specific antigen level significantly related to clinical recurrence after radical retropubic prostatectomy for localized prostate cancer?

OBJECTIVE: To evaluate the influence of preoperative serum prostate-specific antigen (PSA) level and other clinicopathological variables on the probability of biochemical failure and clinical recurrence after radical prostatectomy (RP) for localized prostate cancer. PATIENTS AND METHODS: The study was a retrospective survival analysis in 211 patients undergoing retropubic RP for clinically localized prostate cancer in the period 1988-2000. Survival was estimated using the Kaplan-Meier method; survival endpoints were biochemical failure, defined as a PSA level of > or = 0.5 ng/mL or clinical recurrence consisting of palpable tumours in the prostatic fossa or distant metastases. In 58 patients with biochemical failure after surgery, we assessed the impact of the doubling time of serum PSA level (PSADT) on the risk of developing skeletal metastases or local recurrence. RESULTS: The median (range) observation period was 66 (9-160) months. Biochemical failure occurred in 92 patients (44%) of whom 39 (42%) had local recurrence or skeletal metastases. There was a highly significant association (P < 0.001) between clinical T stage, histological grade, capsular penetration, surgical margin status, seminal vesicle invasion, preoperative serum PSA level and the probability of biochemical failure-free survival. By contrast there was no statistically significant association between preoperative serum PSA level, clinical T stage, surgical margin status, and clinical recurrence. There was a significant relationship between age (P = 0.021), histological grade (P = 0.025), capsular penetration (P = 0.018), seminal vesicle invasion (P = 0014), and clinical recurrence. Cox regression analysis showed that only histological grade and seminal vesicle invasion were independent predictors of clinical recurrence. In a subgroup of 58 patients with a rising serum PSA level after RP, a PSADT of < or = 12.8 months conferred a significantly higher risk (P = 0.015) of developing skeletal metastases than a PSADT of >12.8 months. CONCLUSION: In the present patients undergoing RP the preoperative serum PSA level was not associated with the clinical outcome, whereas it was significantly related to biochemical failure rate. The probability of skeletal metastases was significantly associated with the PSADT after biochemical failure.

The in vitro effect of paclitaxel on a LacZ-transfected malignant transitional cell line.

As bladder cancer is potentially lethal, the development of effective and tolerable therapeutic options is vital. In the present assay, we examined the in vitro effect of paclitaxel (Taxol) on the transitional cell carcinoma (TCC) cell line Hu1703He. Our model has several advantages over other in vitro models. The microenvironment in vivo is mimicked, and the important interaction between benign and malignant cells is consequently preserved in vitro. In addition, the results are not influenced by humoral immune factors. LacZ transfection and exposure to X-gal resulted in blue staining of the tumour cells and made them easy to visualise in sections. Tumour cell aggregates were cultured with continuous paclitaxel exposure to examine the drug's effect on tumour cell migration in monolayer and spheroidal growth in suspension culture. Paclitaxel treatment inhibited both tumour cell migration and spheroidal growth. Invasion was studied by confronting paclitaxel-treated and untreated tumour spheroids with benign bladder fragments in suspension culture. The co-cultures were followed for 4 weeks. Growth of the tumour cells encircling the bladder fragment and cellular infiltration of the bladder stroma were both inhibited by paclitaxel treatment. The expression of MMP-1 in tumour cells was also negatively influenced.

Nonadhesive organ culture of human exocrine pancreatic cells with their stroma.

INTRODUCTION: Studies using explanted tissue have shown that it is possible to keep adult human cells in organ culture with a preserved morphology for up to 1 month as spheres in a nonadhesive organ culture. AIMS: The current study was to determine whether human exocrine pancreatic cells also can be grown in this manner. METHODOLOGY: Small tissue samples from organ donors and tumor-free resection rim from patients with pancreatic carcinoma were obtained (n = 16 adults). From each patient, fragments of approximately 300 microm in diameter were cultured and investigated with light microscopy and scanning and transmission electron microscopy at the time of explantation and after 5, 10, 20, 30, and 40 days of culture. RESULTS: Incubation of cultured fragments with vital dyes revealed a viable epithelium. At the time of explantation all the tissue fragments had a rough appearance with an uneven, torn periphery. During the first week of culture the fragments became rounder, with a smooth surface covering the whole circumference. This spheroid morphology persisted for the rest of the 6-week culture period. The fragments were within 1 week covered by a highly differentiated, polarized epithelium with secretory apparatus, apical secretion granules, and microvilli, as well as specialized cell junctions, with the same appearance as acinoductal pancreatic cells of the original tissue. The core of the fragments consisted of connective tissue with vascular elements, fibroblasts, leukocytes, and a few ductal and acinar elements. Transmission electron microscopy of the spheroids revealed a continuous basal lamina underneath the epithelium. Immunostaining for cytokeratin 5, 6, 7, 8, 17, and 18 was strongly positive in the epithelium. CONCLUSION: These results show that normal exocrine pancreatic cells can be grown in vitro in a nonadhesive organ culture with their stroma.