Section 6 -

Molecular Alterations Associated with Bladder Cancer Initiation and Progression Carlos Cordon-Cardo, M.D., Ph.D. Department of Pathology, Division of Molecular Pathology Memorial Sloan-Kettering Cancer Center New York, N.Y.

Bladder cancer: Clinicopathological characteristics and economical impact. Bladder cancer is one of the most common malignancies occurring worldwide, and is a major cause of morbidity and mortality. The prevalence of bladder cancer is highest in developed countries. It is the fifth most commonly diagnosed non-cutaneous solid malignancy, and the second most commonly diagnosed genitourinary malignancy amongst people living in the United States [1, 2] where it is estimated that more than 60,000 new cases of bladder cancer have been diagnosed in the year 2005 [2]. Bladder cancer poses a significant economic burden to the U.S. health care system. Unlike other common malignancies, bladder cancer is characterized by frequent recurrences, requiring intensive surveillance with office cystoscopies and urinary cytologies, as well as frequent tumor resections under anesthesia. Such physician dependent surveillance procedures, combined with the often long natural history of superficial bladder tumors, make bladder cancer the most costly malignancy to the Medicare system (Figure 1) [3, 4]. Figure 1 Approximately 90% of malignant tumors arising in the urinary bladder are of epithelial origin, the majority being transitional cell carcinomas [1, 5]. Early stage bladder tumors have been classified into two groups with distinct behavior and unique molecular profiles: low grade tumors (always papillary and usually superficial), and high-grade tumors (either papillary or non-papillary, and often invasive} (see Figure 2} [5, 6, 7]. Clinically, superficial bladder tumors (stages Ta, Tis, and T1) account for 75% to 85% of neoplasms, while the remaining 15% to 25% are invasive (T2, T3, T4) or metastatic lesions at the time of initial presentation. Metastatic bladder cancer is an important focus of research because, in most cases, cancer-related death is the result of systemic disease rather than the primary tumor. Even with recent improvements in primary therapy and systemic chemotherapy, the median survival for patients with unresectable metastatic bladder cancer is approximately 7-20 months. Since criteria to determine treatment in a particular patient are incompletely defined, new biological determinants are needed for proper selection and monitoring of therapy. Need for disease specific bladder cancer progression biomarkers. Studies from our group and others have revealed that distinct genotypic and phenotypic patterns are associated with early versus late stages of bladder cancer. Most importantly, early superficial diseases appear to segregate into two main pathways. Briefly, chromosome 9 deletions and mutations of RAS and FGFR3 are observed in most if not all superficial papillary non-invasive tumors (Ta), but only in a small subset of invasive bladder neoplasms. However, deletions of 3p, 5q, 10q (PTEN locus), 11p, 13q (RB locus), 17p (TP53 locus), and 18q (DCC locus) are absent or very rare in the Ta tumors analyzed, but have been frequently identified in invasive bladder carcinomas. Based on these data, a novel model for bladder tumor progression has been proposed in which two separate genetic pathways characterize the evolution of superficial bladder neoplasms [5, 6, 7] (Figure 2 – see below for details in molecular alterations). Figure 2 Bladder tumors are initially identified by cystoscopy. Transurethral resection (removal of tumor from the bladder through a scope inserted transurethrally) is performed in an attempt for cure, as well as tissue diagnosis. Intravesical bacille Calmette-Guerin (BCG) represents one of the main treatments for carcinoma in situ or high-grade superficial bladder carcinoma. Muscle infiltrating tumors are generally treated by cystectomy with or without adjuvant chemotherapy. Radical cystectomy includes total removal of the bladder and surrounding tissues. While it is known that a complete lymph node dissection has an important impact on pathologic staging and outcomes, significant variation exists in how the operation is performed leading to significant clinical bias. At our institution, an ongoing study is testing the benefits of extended lymph node dissection over a standard lymph node dissection. This approach includes resection of lymph nodes below and above the common iliac bifurcation. After surgery, all patients with advanced disease are offered adjuvant chemotherapy, usually with either Gemcitabine and Cisplatin or Gemcitabine and Carboplatin. As stated above, the diagnosis and follow-up of patients with bladder cancer is based on the information provided by cystoscopy in combination with urinary cytology. Many urinary tumor markers have been evaluated for the detection and surveillance of the disease, providing promising results as complementary tests to cytology. Hovewer, they are neither fully validated nor introduced in clinical practice yet [8]. In serum, none of the tumor biomarkers evaluated to date has provided sufficient sensitivity and specificity for the early detection of superficial bladder cancer, nor favorable efficacy for predicting relapses, response to chemotherapy, and overall survival in patients with advanced disease. Bladder cancer prognostication is based on pathological stage and grade, tumor size, presence of concomitant carcinoma in situ, and multicentricity. Lymphovascular and/or perineural invasion, as well as squamous differentiation, are considered poor prognosticators in bladder cancer. Numerous individual molecular markers have been identified in the tissue specimens that correlate to some extent with tumor stage, and possibly with prognosis in bladder cancer. However, these molecular prognosticators do not play a role in the clinical routine management of patients with bladder tumors, mainly due to lack of large prospective validation studies. Thus, the need for development of specific tissue and serum tumor markers for prognostic stratification remains. The advent of high-throughput microarrays technologies allows comprehensive discovery of targets relevant in bladder cancer progression, which could be translated into new approaches for drug and biomarker development. Further investigation is warranted to define novel biomarkers specific for bladder cancer patients based on the molecular alterations of tumor progression, and multiplexed strategies for clinical management. Oncogenes and tumor suppressor genes in bladder cancer progression. The human RAS genes represent a family of cellular transforming oncogenes, H-RAS originally identified in the human urothelial cell line T24 [9]. Two mechanisms have been reported for gene transformation. The most frequent is mutations affecting the enzymatic activity of the encoded Ras protein, more frequently found on codons 12, 13, 59 and 61. For example, mutations in the coding sequence of the H-RAS at codon 12 are reported to occur in around 30-40% of urothelial malignancies [10]. A second mechanism is due to internal splicing within the last intron affecting RAS gene expression. Concurrent mutations within the splicing mechanism and the coding sequence resulting in the overexpression of the transforming gene product are found in high grade and high stage tumors at a low prevalence, lower than 10%. Although several groups have evaluated RAS genes in bladder cancer, their biological and clinical significance remains to be elucidated. Even though several growth factor receptors, such the epidermal growth factor receptor (EGFR), have been found over-expressed in bladder cancer, only the fibroblast growth factor receptor 3 (FGFR3) gene has been reported to be mutated, specifically in superficial bladder tumors [7]. Activation of this tyrosine kinase by any of the numerous identified ligands, or mutation, results in enhanced cell growth as well as angiogenesis. It has also been reported that FGFR3 and H-RAS mutations are mutually exclusive, since they are involved in the same signaling pathway, the so-called RAS-MEK-ERK network. Up to around 75% of all Ta lesions harbor FGFR3 mutations, but alterations of this gene have not been found in Tis tumors, further supporting the multiple nature of molecular pathways to bladder tumorigenesis. Alterations in both p53 and RB tumor suppressor gene pathways are well documented for bladder cancer. The most frequent alterations are mutations in exons 5 to 11 of TP53, and the loss of pRB gene function [11, 12, 13, 14, 15]. This mechanism was identified in approximately 60% of human urinary bladder cancer cell lines tested. In addition, around 20% of analyzed cell lines harbor an independent synchronous loss of the tandemly linked CDKN2A (p16) and ARF (p19) genes in at 9p21. This results in transformation and immortalization of urothelial cells, by indirect impairing of both p53 (Arf) and RB (p16) pathways. In a similar 20% percentage of cell lines, p53 was found to be mutated in exons 1-4 followed by loss of p16 and p19Arf function. Thus, patterns of p53 mutations may be a prerequisite for subsequent, distinctive molecular events on other genes such as RB, as well as CDKN2A and ARF [15]. Interestingly, in an independent study, gene profiling of bladder cancer cell lines supporting the relevance of such combined alterations in the stratification of the cell lines and their transcript profiles [16]. Identification of molecular targets involved in bladder cancer progression using microarray technologies. The advent of high-throughput methods of molecular analysis has allowed the comprehensive survey of the genetic profiles characteristic of distinct tumor types, and identify targets and pathways that may underlie particular clinical behavior. Several groups have also used gene expression profiling of bladder cancer tissues to identify signature genes that robustly distinguish bladder cancer subclasses, and genetic pathways underlying bladder cancer progression [17, 18, 19, 20, 21]. However, further research is warranted in the field to translate the identification of these molecular targets into potential predictive biomarkers of bladder cancer behavior. The challenge remains to optimize the measurement of these targets on non-invasive specimens and to improve outcome stratification, finally impacting on clinical management. Microarrays represent a convenient platform for assays involving biomolecules other than nucleic acids [22]. Arrays of peptides, antibodies, proteins, and even cells have been developed [22, 23]. This is further evidence of the strength and versatility for high throughput screening. These should provide means of rapidly validating at the protein level, the genes identified by expression profiling using DNA microarrays. Comparative fluorescence can not only measure the relative abundance of genetic sequences, but also estimate many antigens once specific antibody solutions are printed on the surface of derivatized surfaces. Antibody and protein microarrays have been applied to protein profiling of cancer tissue or antibody-based detection of multiple antigens [22, 23]. Toward the development of such capability, it is possible to use a practical strategy for the use of antibody microarrays for highly parallel screening of potential progression biomarkers in human serum. Very recently the use of protein profiling using antibody arrays for bladder cancer on serum specimens has been described [24]. Such study reports how antibody arrays can utilize the information provided by expression profiling to design targeted antibody arrays for detecting specific clinical behaviors. The highly efficient protein detection method will lead to the discovery of new and clinically useful protein biomarkers of outcome prediction for bladder cancer patients [24]. The advent of high-throughput technologies is allowing comprehensive identification of molecular targets and biomarkers specific for bladder cancer. Such identification process allows a better understanding of the biology associated with tumorigenesis and tumor progression. The challenge remains in evaluating the impact of such targets for therapeutics development and translating progression and outcome biomarkers to improve the clinical management of the bladder cancer patients. Integrative efforts of the complementary message obtained through the diverse technologies at the DNA, RNA and protein level together with multi-institutional validation collaboration studies would result in targeted and tailored therapies based on the aggressiveness of each specific bladder tumor. References Dinney CP, McConkey DJ, Millikan RE, et al. Focus on bladder cancer. Cancer Cell 6:111-116, 2004. Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA Cancer J Clin.55:10-30, 2005. Botteman MF, Pashos CL, Redaelli A, Laskin B, Hauser R. The health economics of bladder cancer: a comprehensive review of the published literature. Pharmacoeconomics 21:1315-1330, 2003. Mitra N, Indurkhya A. 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