Timely diagnosis can help improve cancer control and the quality of life for these patients. Stage, tumor grade, size, multifocality of transitional cell carcinoma (TCC), presence of carcinoma in situ, and lymph node status are currently the most useful findings for making therapeutic decisions and evaluating the prognosis for patients with bladder cancer. However, the remarkable difference in biologic behavior of tumors within the same stage and grade category precludes accurate prediction in an individual patient. Numerous efforts in searching for new, more powerful biomarkers have thus been made.

In the article in this issue, Schrier et al. [1] attempted to establish the predictive value of p53, p27kip1, and a-catenin in patients with progression from superficial to muscle-invasive bladder carcinoma. Forty-one patients with progression after primary superficial bladder carcinoma were individually matched to patients with nonprogressive tumors. They confirmed that expression of p53 protein and a-catenin in primary bladder tumors possesses prognostic value. Combinations of markers had no synergistic effect. Alterations in the genetic material encoding for cell cycle regulators known as tumor suppressor genes are commonly demonstrated in variety of urologic cancers. Esrig et al. [2] are credited as being the first to find p53 changes as predictive for outcome in patients with bladder cancer undergoing cystectomy. An accumulation of p53 in the tumor cell nuclei detected by immunohistochemical methods predicts a significantly increased risk of recurrence and death, independently of tumor grade, stage, and lymph node status. p53 has since been the most widely investigated molecular marker in bladder cancer and, thus, believed to be the most promising to predict the clinical outcome. Immunohistochemistry outcome is, however, bedeviled by its inconsistency. A meta-analysis using 168 publications from 117 studies showed that changes in p53 were only weakly predictive of recurrence, progression, and mortality in bladder cancer [3].

Other biomarkers to predict recurrence and progression of the disease, response to treatment, and survival include oncogenes (c-myc, c-erb-B2, H-ras, and MDM2), chromosomal alternations (chromosome 9, 13, and 17), cell cycle regulatory proteins (pRb, p53 protein, p21, and p27), proliferation-associated antigens (Ki67, PCNA, and MCMs), angiogenic factors (fibroblast growth factor, endothelial growth factor, vascular endothelial growth factor, interleukins, and CD34), cell adhesion molecules (E-cadherin, p120, CD44, ß- and ?-catenin, integrins, and coxsackie and adenovirus receptor), cytokeratins, telomerase, survivin, HSP70, matrix metalloproteinases, nuclear matrix protein 22, hyaluronic acid, and hyaluronidase.

Several investigators have focused on tissue-specific instead of cancer-specific markers, such as prostate-specific antigen in prostate cancer. Among these, uroplakins may be of interest. The apical surface of bladder epithelium is covered with numerous rigid-looking plaques that consist of two-dimensional crystals of hexagonally arranged 16-nm particle-protruding tubules. The apical membrane is almost twice as thick as the inner leaflet of membrane, thus leading to the term, asymmetric unit membrane (AUM). AUMs contain four major integral membrane proteins, uroplakin Ia, Ib, II, and III. Recently, uroplakins have been noted as tissue-specific markers of urinary epithelia. Uroplakin III was found not only in normal epithelia but also in about 60% of primary TCCs as well as metastases. Uroplakins may thus help in detecting micro-metastases. Wu et al. [4] reported 100% of primary bladder cancers and 93.6% of pathologically proven metastases were positive for uroplakin II mRNA. Uroplakin II gene expression was also detected in the peripheral blood from every patient with metastatic TCC but not from patients with nonmetastatic TCC nor healthy volunteers [5]. Although the potential of uroplakins to predict recurrence or prognosis is as yet unknown, they may prove to be one of the most promising candidate markers of progression in bladder cancer.

To date, no single marker has been discovered that provides complete accuracy in predicting the biologic behavior of bladder cancer. More global assessment with molecular profiling or proteomics (or both) rather than single-marker research may be more profitable. High-throughput cDNA and tissue microarrays are promising venues in the field of urologic oncology. Sanchez-Carbayo et al. [6] have used cDNA microarrays containing 17,842 known genes to allow hierarchical clustering of early-stage and invasive TCC. It was possible to distinguish carcinoma in situ from papillary superficial lesions and subgroups within early-stage and invasive tumors displaying different overall survival. Cytokeratin 20, neuropilin-2, p21, and p33ING1 were selected among the top-ranked molecular targets differentially expressed and validated by immunohistochemistry using tissue microarrays. p33ING1 expression levels and overexpression of oncogenic genes such as DEK in superficial tumors were significantly associated with progression and overall survival.

Proteomics, a new field that exhaustively studies protein expression and function in a cell or tissue, appears to be a promising way to better understand the biology of organisms [7]. The proteomic approach is conceptually attractive because it directly elucidates protein expression profiles in tissues, which mRNA levels do only indirectly. Properties studied by the proteomic approach are not limited to the amounts of proteins present, but also include their subcellular location, state of modification, and association with other proteins or ligands. The proteome dynamically depends on the state of the organism, so proteomic studies also demonstrate subtle changes in those properties over time. Cancer proteomics is expected to find new biomarkers, present in serum and other biologic fluids that may assist in early diagnosis of cancer. Tumor-specific proteome profiles also can correlate with clinical features and tumor behavior. Proteomics without electrophoresis has also been applied.

Researchers have discovered new biomarkers using developing proteomic analysis in bladder cancer. Urine calreticulin, ?-synuclein, and catechol-o-methyltransferase are novel biomarkers that were found with high-resolution two-dimensional gel electrophoresis in combination with microsequencing and mass spectrometry [8]. Although these urine marker proteins are merely diagnostic for early-stage TCC at present, some of them may prove to be prognostic in the future. In tumor tissue proteomics, Celis et al. [9] compared protein expression profiles of nonmalignant bladder urothelium with those in TCC using two-dimensional gel electrophoresis and mass spectrometry. Four proteins that were expressed by the nonmalignant urothelium but lost at various stages of bladder cancer progression were identified: GST-µ, prostaglandin dehydrogenase, keratin 13, and the adipocyte-type fatty acid-binding protein (A-FABP). Among these, A-FABP expression significantly correlated with both tumor stage and grade; A-FABP could thus be a promising candidate as a prognostic biomarker in bladder cancer [10]. The ProteinChip technology coupled with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) also facilitates protein profiling of complex biologic mixtures. Multiple protein changes have been reproducibly detected in the urine of patients with TCC.

The application of advanced technology has allowed more profound understanding of the carcinogenesis and provided tools for an extensive search for new candidate biomarkers for biologic potential. The panel of markers and the use of these together with conventional parameters as a multiplex marker model may be more fruitful. Such prediction models need to be validated in large and prospective studies. Progressive genetic and protein profiles on a large number of patients to compare and determine the associations between early- and late-stage patterns are also needed. Research will continue to identify patients who need a more intensive treatment and subsequently to prolong overall survival of patients with bladder cancer. The search for the "holy grail" in urologic oncology still continues.

References

1. Schrier BPh, Vriesema JLJ, Witjes JA, Kiemeney LALM, Schalken JA. The predictive value of p53, p27Kip1, and a-catenin for progression in superficial bladder carcinoma. Eur Urol. 2006;50:76–82.
2. Esrig D, Elmajian D, Groshen S, et al.. Accumulation of nuclear p53 and tumor progression in bladder cancer. N Engl J Med. 1994;331:1259–1264.
3. Malats N, Bustos A, Nascimento CM, et al.. p53 as a prognostic marker for bladder cancer: a meta-analysis and review. Lancet Oncol. 2005;6:678–686.
4. Wu X, Kakehi Y, Zeng Y, Taoka R, Tsunemori H, Inui M. Uroplakin II as a promising marker for molecular diagnosis of nodal metastases from bladder cancer: comparison with cytokeratin 20. J Urol. 2005;174:2138–2142.
5. Yuasa T, Yoshiki T, Isono T, Tanaka T, Hayashida H, Okada Y. Expression of transitional cell-specific genes, uroplakin Ia and II, in bladder cancer: detection of circulating cancer cells in the peripheral blood of metastatic patients. Int J Urol. 1999;6:286–292.
6. Sanchez-Carbayo M, Socci ND, Lozano JJ, et al.. Gene discovery in bladder cancer progression using cDNA microarrays. Am J Pathol. 2003;163:505–516.
7. Kuruma H, Egawa S, Oh-Ishi M, et al.. High molecular mass proteome of androgen-independent prostate cancer. Proteomics. 2005;5:1097–1112.
8. Iwaki H, Kageyama S, Isono T, et al.. Diagnostic potential in bladder cancer of a panel of tumor markers (calreticulin, ?-synuclein, and catechol-o-methyltransferase) identified by proteomic analysis. Cancer Sci. 2004;95:955–961.
9. Celis JE, Ostergaard M, Basse B, et al.. Loss of adipocyte-type fatty acid binding protein and other protein biomarkers is associated with progression of human bladder transitional cell carcinomas. Cancer Res. 1996;56:4782–4790.
10. Ohlsson G, Moreira JM, Gromov P, Sauter G, Celis JE. Loss of expression of the adipocyte-type fatty acid-binding protein (A-FABP) is associated with progression of human urothelial carcinomas. Mol Cell Proteomics. 2005;4:570–581.

Shin Egawa, Hidetoshi Kuruma,
Department of Urology, Jikei University School of Medicine, Tokyo, Japan

published online 27 February 2006.

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