High expression of Wnt7b in human superficial bladder cancer vs invasive bladder cancer.

Aberrant Wnt gene expression is involved in the development of breast cancer, but its role in other tumours is unknown. Wnts regulate cadherin function, previously shown to be more commonly deregulated in invasive bladder cancer. This study investigated whether factors upstream of cadherins were aberrantly expressed in superficial bladder cancer. The expression of one transforming (Wnt7b) and one non-transforming (Wnt5a) Wnt gene in four human bladder carcinoma cell lines, and in normal human bladder tissues (n = 8) and bladder cancers (n = 48) were analysed by ribonuclease protection analysis. All cell lines expressed an approximately equal level of Wnt7b mRNA. Wnt5a and Wnt7b mRNAs were both expressed in normal bladder tissues and bladder tumours. The median expression of Wnt7b was fourfold higher in superficial tumours (n = 29) than in normal tissues (n = 8, P = 0.002) and five fold higher than in invasive tumours (n = 17, P = 0.003). There was no significant difference between normal tissues and invasive tumours (P = 0.3). The expression of Wnt5a did not vary significantly between normal tissues and superficial tumours (P = 0.4), normal tissues and invasive tumours (P = 0.3) or superficial tumours and invasive tumours (P = 0.2). The differential expression of Wnt7b suggests a role in the early events of superficial bladder tumorigenesis involving cell adhesion and provides further evidence of different pathways of evolution of superficial and invasive cancer.     

Prognostic factors and therapy for superficial and invasive bladder cancer.

Although most of the transitional cell carcinomas are superficial at presentation and pose little problem as far as survival is concerned, predicting the ones that will recur and, more importantly, recur as invasive tumors, is crucial. Determination of serum and urine human chorionic gonadotropin-beta levels and epidermal growth factor receptor analysis are promising in this respect. Intravesical bacillus Calmette-Guérin, which is widely used to prevent further recurrences and to treat carcinoma in situ, augments the host's immune status, probably by increasing interleukin-2 and tumor necrosis factor levels. Intravesical interferon seems to be a promising agent, especially against carcinoma in situ, and has exceptionally few side effects. Multidrug chemotherapy regimens such as methotrexate, vinblastine, doxorubicin, and cisplatin, or cisplatin, methotrexate, and vinblastine have achieved 30% to 40% objective complete remission rates in patients with metastatic bladder cancer. These regimens are also used in a neoadjuvant setting with or without irradiation in the hope that more patients will be spared cystectomy and enjoy similar if not better survival. At the same time, continent diversions and nerve-sparing cystoprostatectomy open a new era in the treatment of invasive bladder cancer by improving the quality of life after cystectomy. Although important progress has been made, there is still room for research and clinical controlled studies to achieve higher rates of response, prolonged disease-free survival, and perhaps even improved rates of cure.     

Visualizing superficial human bladder cancer cell growth in vivo by green fluorescent protein expression

There has been no reliable orthotopic model available to visualize the growth of human superficial bladder cancer over time and to evaluate the efficacy of intravesical therapies. We have developed a novel approach to accomplish this task by generating human superficial bladder tumor cells to stably express high levels of green fluorescent protein (GFP) in vivo. Superficial bladder tumors were produced in athymic mice by intravesical instillation. In our initial studies tumors were quantitated by image analysis at a single time point, and the results compared to the estimation of the percentage of GFP cells present using flow cytometry after obtaining single cell suspensions of normal and tumor cells in the same bladder. A high correlation between the two methods was seen. Therefore, in subsequent studies, approximately 1 week after the intravesical instillation of the GFP expressing cancer cells a small incision was made to expose the bladder. The anterior, posterior, and lateral images of each bladder were captured to visualize GFP-expressing tumors. The ratio of green fluorescence pixel area, which represented the tumor burden, to the total area of the bladder was then calculated. A similar procedure was performed at 2, 3, and 4 weeks after instillation of the tumor cells. Using this procedure tumor progression over time could be measured in each mouse. By using this approach, it will now be possible to monitor the initial tumor sizes in the bladder of each mouse and then to evaluate the efficacy of various intravesical therapy protocols including intravesical gene therapy alone or in combination with other treatment modalities.     

Differing expression of enzymes of the glyoxalase system in superficial and invasive bladder carcinomas

This work aimed to study the activities of the glyoxalase system enzymes (glyoxalase I (GI) and glyoxalase II (GII) and their gene expression in human bladder carcinomas compared with the corresponding normal mucosa. Samples of these tissues were collected from 26 patients with superficial (SBC) or invasive bladder cancer (IBC) and used to evaluate enzyme activity and gene expression by northern blot analysis. In keeping with the electrophoretic pattern and the expression level of the respective genes, GI activity significantly increased in SBC samples, while it remained unchanged in IBC samples compared with the normal mucosa. In contrast, GII showed a higher activity in the tumour (either SBC or IBC samples) versus normal tissues. These results confirm the role of the glyoxalases in detoxifying cytotoxic methylglyoxal (MG) in bladder cancer. The differing levels of GI activity level and gene expression of GI between the SBC and IBC samples could help in their differential diagnosis.     

Chemoprevention of superficial bladder cancer

The American Cancer Society estimates that 57,400 new cases of bladder cancer will be diagnosed in the USA in 2003 and that approximately 70% of these cases will be superficial bladder tumors. Due to the high risk of recurrence, patients with superficial bladder cancer represent an ideal group for chemoprevention. Intravesical chemotherapy or immunotherapy is often administered in an attempt to prevent tumor recurrence in high-risk patients, although it is not without toxicity. A large body of evidence links diet and nutrition with bladder cancer. This review summarizes the efficacy of natural and synthetic agents that have purported chemopreventive activity.     

Chemoprevention of superficial bladder cancer

The American Cancer Society estimates that 57,400 new cases of bladder cancer will be diagnosed in the USA in 2003 and that approximately 70% of these cases will be superficial bladder tumors. Due to the high risk of recurrence, patients with superficial bladder cancer represent an ideal group for chemoprevention. Intravesical chemotherapy or immunotherapy is often administered in an attempt to prevent tumor recurrence in high-risk patients, although it is not without toxicity. A large body of evidence links diet and nutrition with bladder cancer. This review summarizes the efficacy of natural and synthetic agents that have purported chemopreventive activity.     

Molecular genetic alterations in superficial and locally advanced human bladder cancer.

We attempted to define the role of tumor suppressor genes in the pathogenesis of human bladder cancer through a combined molecular genetic and immunohistochemical approach. Thirty-four bladder tumors (1 Pis, 6 Pa, 5 P1, 3 P3a, 18 P3b, 1 P4; 8 low grade and 26 high grade tumors) have been analyzed. Restriction fragment length polymorphism analysis directed at 5 suspected or established tumor suppressor gene regions (3p21-25, 11p15, 13q14, 17p11-13, and 18q21) was combined with immunohistochemical using Rb-PMG3-245 monoclonal antibody directed at the retinoblastoma (Rb) gene product. Tumor grade correlated with deletions of 3p (P = 0.004) and 17p (P = 0.063). Tumor stage correlated with deletions of 3p (P = 0.010), 17p (P = 0.015) and altered Rb expression (P = 0.054). Vascular invasion correlated only with deletions of 17p (P = 0.038). No marker correlated with positive lymph nodes. Our results suggest that altered Rb expression occurs in all grades and stages of bladder cancer but is more commonly associated with invasive tumors. Genetic alterations of 3p, 11p, 17p, and 18q are rare events in low grade, superficial tumors, whereas they are more common in high grade and invasive bladder cancer. The role of these genetic alterations in the prognosis of bladder cancer will require additional follow-up and further studies.     

Prognostic significance of mutations in the p53 gene in superficial bladder cancer

Bladder tumors as the most common urologic malignancy present mostly as superficial transitional cell carcinoma. Many patients with superficial bladder cancer have a good prognosis, however, may develop recurrences or progress to muscle invasive or metastatic disease. It is therefore important to find new markers associated with the biological behaviour of an individual tumor for identifying patients at risk for disease progression. Previous reports on the prognostic significance of p53 alterations in bladder tumors revealed conflicting results. The aim of our study was to evaluate p53 mutation analysis as an effective concept for the characterization of subsets of superficial bladder tumors differing in biological aggressiveness. Screening 66 amplified DNA from micro-dissected tumor cells by direct genomic sequencing p53 alterations were detected in 12%. We found no association between p53 status and tumor stage but a tendency to a higher mutation rate in more malignant tumors (G2 and G3) compared to G1 tumors and a higher recurrence rate in patients with a p53 mutation in the primary tumor after 24 months follow-up. We conclude a general low incidence of p53 mutations in superficial bladder cancer. Detectable p53 damage might be related to a more aggressive phenotype and a higher recurrence risk. Our results are discussed in the context of other studies reviewed from 1995-2000.     

Diagnosis and management of superficial bladder cancer

Bladder cancer is the fourth leading cause of cancer in American men, accounting for more than 12,000 deaths annually. It was one of the first malignancies in which carcinogens were recognized as an important factor in its cause. Currently, cigarette smoking is by far the most common cause of bladder cancer, although occupational exposure to arylamines has been implicated in the past. Gross or microscopic hematuria is the most common sign at presentation. Initial radiologic evaluation usually includes the excretory urography (intravenous pyelography), although further evaluation of the renal parenchyma with ultrasound or computed tomography scanning has been advocated by some. These radiologic studies are unable to provide adequate bladder imaging, and thus cystoscopy is required for the diagnosis of bladder cancer. Most bladder cancers present as "superficial" disease, confined to the bladder mucosa or submucosal layer, without muscle invasion. Superficial tumors consist of papillary tumors that are mucosally confined (Ta), papillary or sessile tumors extending into the lamina propria (T1), and carcinoma in situ, which occurs as "flat" mucosal dysplasia, which can be focal, diffuse, or associated with a papillary or sessile tumor. The natural history of these pathologic subtypes differ significantly. Most superficial tumors (60% to 70%) have a propensity for recurrence after transurethral resection. Some (15% to 25%) are at high risk for progression to muscle invasion. Most superficial tumors can be stratified into high- or low-risk groups depending on tumor stage, grade, size, number, and recurrence pattern. It is important to identify those tumors at risk for recurrence or progression so that adjuvant intravesical therapies can be instituted. Many intravesical chemotherapeutic agents have been shown to reduce tumor recurrence when used in conjunction with transurethral tumor resection. Unfortunately, however, none of these agents have proved to be of benefit in preventing disease progression. Most are given intravesically on a weekly basis, although many studies suggest that a single instillation immediately after transurethral resection may be as good as a longer course of therapy. Although all of these drugs have toxicity, they usually are well tolerated. Intravesical bacille Calmette-Guérin (BCG) is an immunotherapeutic agent that when given intravesically is very effective in the treatment of superficial transitional cell carcinoma. Compared with controls, BCG has a 43% advantage in preventing tumor recurrence, a significantly better rate than the 16% to 21% advantage of intravesical chemotherapy. In addition, BCG is particularly effective in the treatment of carcinoma in situ, eradicating it in more than 80% of cases. In contrast to intravesical chemotherapy, BCG has also been shown to decrease the risk of tumor progression. The optimal course of BCG appears to be a 6-week course of weekly instillations, followed by a 3-week course at 3 months in those tumors that do not respond. In high-risk cancers, maintenance BCG administered for 3 weeks every 6 months may be optimal in limiting recurrence and preventing progression. Unfortunately, adverse effects associated with this prolonged therapy may limit its widespread applicability. In those patients at high risk in whom BCG therapy fails, intravesical interferon-alpha with or without BCG may be beneficial in some. Photodynamic therapy has also been used but is limited by its toxicity. In patients who progress or do not respond to intravesical therapies, cystectomy should be considered. With the development of orthotopic lower urinary tract reconstruction to the native urethra, the quality of life impact of radical cystectomy has been lessened.     

Diagnosis and management of superficial bladder cancer

Superficial transitional cell carcinoma is defined as a transitional cell urothelial tumor that is confined to the mucosa, stages Ta or CIS, or with invasion of the lamina propria, T1. The initial treatment is transurethral resection with an attempt to remove all tumor. This should provide an accurate histologic grade and stage, and from this information a prognosis can be determined. The important predictive factors that correlate with a new occurrence or true recurrence and the development of a subsequent tumor with muscle invasion are a high tumor grade, lamina propria invasion, a positive cytology following resection, multifocal tumors, dysplasia or carcinoma in situ from mucosal biopsies of normal appearing urothelium, and a prior history of bladder cancer. Based on these factors, the recurrence rate varies from 30 to 80% and progression with a muscle invasive tumor up to 30%. Intravesical chemotherapy or "immunotherapy" following tumor resection has been shown to diminish the likelihood of a recurrence. Thiotepa has been used for the longest period of time. It is relatively inexpensive, safe if myelosuppression is closely monitored, and effective. Mitomycin C was more effective than Thiotepa in randomized trials, but is significantly more expensive. This has retarded its use as a first-line agent. It has been shown to eradicate persistent tumor in 30 to 40% of patients who have failed Thiotepa. Mitomycin C is also highly effective when used for prophylaxis. Intravesical bacillus Calmette-Guerin (BCG) has recently been demonstrated to be an effective intravesical therapeutic agent. It is effective both for treatment and prophylaxis. BCG is relatively safe and inexpensive.(ABSTRACT TRUNCATED AT 250 WORDS)     

The hypoxia-inducible genes VEGF and CA9 are differentially regulated in superficial vs invasive bladder cancer

Regulation by hypoxia may underlie the expression of vascular endothelial growth factor in bladder cancer. We have compared the distribution of vascular endothelial growth factor mRNA with a hypoxia marker, carbonic anhydrase 9 (CA IX). vascular endothelial growth factor mRNA was analysed by in situ hybridisation and CA IX by immunochemistry in 22 cases of bladder cancer. The relationship of microvessels to the distribution of CA IX was determined. In a separate series of 49 superficial tumours, CA IX immunostaining was compared with clinico-pathological outcome. In superficial and invasive disease there was overlap in the expression of vascular endothelial growth factor and CA IX, CA IX being more widespread. Both were expressed predominantly on the luminal surface, and surrounding areas of necrosis (invasive tumours). Expression of both factors was greater in superficial disease. Expression was absent within approximately 80 microm of microvessels. Unlike vascular endothelial growth factor, CA IX did not predict outcome in superficial disease. Differential responses to reoxygenation provide one explanation: vascular endothelial growth factor mRNA declined rapidly, while CA IX expression was sustained for >72 h. Expression of vascular endothelial growth factor mRNA in bladder tumours is consistent with hypoxic regulation and suggests differential regulation in superficial vs invasive disease. The expression of CA IX on the luminal surface justifies investigation of its utility as a therapeutic target/prognostic indicator.     

非肌层浸润性和肌层浸润性膀胱癌的双向电泳图谱的差异分析

背景与目的:膀胱癌是泌尿系统最常见的一种恶性肿瘤,不同分期的膀胱癌患者治疗和预后有明显不同.本研究旨在利用蛋白质组学的方法建立非肌层浸润性膀胱癌(non-muscle-invasive bladder cancer)和肌层浸润性膀胱癌(invasive bladder cancer)的差异蛋白质表达谱,并鉴定获得相关蛋白.方法:对30例人非肌层浸润性膀胱癌组织及20例肌层浸润性膀胱癌组织进行比较蛋白质组学研究.即利用双向凝胶电泳(two-dimensional electrophoresiS,2-DE)分离二者总蛋白质后,经图像分析识别差异表达的蛋白,应用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flightmass spectrometry,MRLDI-TOF-MS)鉴定差异蛋白质.结果:比较分析了非肌层浸润性膀胱癌及肌层浸润性膀胱癌组织的2-DE图谱,找到差异蛋白质点,对60个差异蛋白质点进行了肽质量指纹图分析. 结论:成功鉴定了43个非肌层浸润性膀胱癌组织和肌层浸润性膀胱癌组织的差异蛋白,其中一些与肿瘤的生长、黏附、凋亡以及浸润转移相关,为进一步揭示膀胱癌进展的分子机制奠定了基础.     

Prognostic and functional significance of thromboxane synthase gene overexpression in invasive bladder cancer

Thromboxane synthase (TXAS) is one of the enzymes downstream from cyclooxygenase-2 and catalyzes the synthesis of thromboxane A(2) (TXA(2)). TXAS was among the genes we identified based on its overexpression in invasive bladder tumors. TXAS is overexpressed in common forms of bladder tumors: 69 of 97 (71.1%) transitional cell carcinoma (TCC), 38 of 53 (71.6%) squamous cell carcinoma, and 5 of 11 (45.5%) adenocarcinoma relative to nontumor tissue. Overall, 112 of 161 (69.5%) invasive tumors exhibited elevated expression. Significantly, patients with tumors having >4-fold levels of TXAS expression showed significant statistical evidence of lower overall survival expressed by the estimated hazard ratio of 2.74 with P = 0.009 in Cox's regression analysis. TXAS mRNA expression was found to be an independent prognostic marker for patients with bladder cancer. Treatment of bladder cancer cell lines (T24 and TCC-SUP) with TXAS inhibitors and TXA(2) (TP) receptor antagonists reduced cell growth, migration, and invasion, whereas TP agonists stimulated cell migration and invasion. The positive correlation between elevated TXAS expression and shorter patient survival supports a potential role for TXAS-regulated pathways in tumor invasion and metastases and suggests that modulation of the TXAS pathway may offer a novel therapeutic approach.     

Intravesical therapy for superficial bladder cancer

Approximately 54,400 new cases of transitional cell carcinoma of the bladder were reported in the United States in 1999, with an estimated 12,500 deaths attributable to this cancer. Close to 75% of all bladder tumors are confined to the urothelium (stage Ta, or carcinoma in situ), and nearly 30% of papillary tumors invade the lamina propria (stage T1). The majority of superficial tumors are low grade with low rates of progression. Transurethral resection is the standard initial treatment for transitional cell carcinoma. Intravesical therapy is an important adjunct to transurethral resection in patients with superficial bladder cancer, many of whom are at risk for disease recurrence and progression. Cytotoxic and immunomodulating agents and, more recently, photosensitizers have demonstrated utility against superficial transitional cell carcinoma. Many studies have assessed and continue to examine the efficacy of various agents at different doses and in different combinations and schedules. Recently, valrubicin (Valstar) won Food and Drug Administration (FDA) approval only for the treatment of refractory carcinoma in situ. However, bacillus Calmette-Guérin (BCG) and mitomycin (Mutamycin) remain the most commonly used, most effective agents available for prophylaxis against recurrence and subsequent progression of superficial bladder cancer. This article reviews traditional and alternative intravesical agents useful in the therapy and prophylaxis of superficial transitional cell carcinoma of the bladder.