Predicting Prognosis in Patients With Superficial Bladder Cance Published on Cancer Network (http://www.cancernetwork.com)

Predicting Prognosis in Patients With Superficial Bladder Cancer Review Article [1] | December 01, 1998 | Genitourinary Cancers [2], Bladder Cancer [3] By Ralph W. Devere White, MD [4] and Eschelle Stapp, MD [5] Bladder cancer is the most common urologic malignancy and is expected to affect approximately 54,000 people in 1998. Superficial bladder tumors (Tis, Ta, and T1 lesions) account for approximately 70% to 80% of these

Introduction ladder cancer is the most common malignancy encountered by urologists. It is estimated that, in 1998, 54,400 cases of bladder cancer will be diagnosed in the United States, leading to approximately 12,500 deaths.[1] B

More than 90% of bladder tumors are transitional cell carcinomas. Risk factors for the development transitional cell carcinoma include cigarette smoking and exposure to arylamines (such as aniline dyes), certain drugs, and pelvic radiation.[2-5] Cigarette smoking is the cause of 25% to 60% of all bladder cancers, and smokers have two times the risk of developing this cancer than do nonsmokers.[5] Fortunately, most patients (80%) with newly diagnosed bladder cancer present with “superficial” disease, which can be managed with transurethral resection or intravesical therapy. The term “superficial bladder cancer” refers to tumors confined to the mucosa (Ta), those with lamina propria invasion (T1), and carcinoma in situ (Tis).[6] The recurrence rate for superficial bladder tumors is high (two-thirds), and, despite advances in management, some patients still develop stage progression. It is well known that such stage progression occurs more often in patients with high-grade lesions and T1 tumors. For this reason, it seems imprecise to group all “superficial” tumors together. Tumors that pose a high risk for progression and death are multifocal Ta lesions, tumors with associated Tis disease, and T1 lesions. Eventually, one-half of these high-risk patients will require a cystectomy, and one-third are at risk of dying from bladder cancer over 15 to 20 years.[6] A prognostic marker that could identify individuals at risk for recurrence and progression might dramatically change the clinical management of patients with bladder cancer. The availability of such a marker would permit the selective use of aggressive treatment in patients who are at high risk of recurrence and progression while sparing low-risk patients from un-necessary procedures.[7] Researchers are investigating several prognostic factors that may provide such a model. Traditional prognostic factors in bladder cancer have included tumor characteristics based on cystoscopic and pathologic findings. Certain biological markers may also play important roles in predicting high-risk tumors. These include DNA ploidy, S-phase, certain monoclonal antibodies, the p53 (alias TP53) tumor-suppressor gene, the retinoblastoma (Rb) gene, cell adhesion molecules, and angiogenesis.

Tumor Characteristics Cystoscopic Findings Characteristics of bladder tumors that are visible with cystoscopy can have important prognostic implications. For example, tumor size may play a role in progression to muscle invasive disease. In one series, tumors > 5 cm had a 35% progression rate, as compared with a 9% rate for smaller Page 1 of 10

Predicting Prognosis in Patients With Superficial Bladder Cance Published on Cancer Network (http://www.cancernetwork.com) tumors.[8] The number of tumors is also important for predicting recurrence but not progression.[8] Recurrence rates in patients with single tumors range from 18% to 60%, as opposed to rates of 40% to 90% in those with multiple tumors.[6] The timing of first recurrence is another significant cystoscopic finding. Fitzpatrick et al reported that, among patients with Ta lesions who had no recurrences at their first post-resection cystoscopy, 79% had no subsequent recurrences.[9] In contrast, 90% of patients with recurrences at 3 months also had additional recurrences. Lastly, tumor appearance or morphology can also be prognostic, with sessile (solid) masses having a higher recurrence rate than papillary masses.[9] Pathologic Findings Pathologic characteristics of superficial bladder tumors that have the greatest prognostic significance are tumor stage and grade. The most commonly used staging system for transitional cell carcinoma is the tumor, node, metastasis (TNM) classification, which has recently been updated (Table 1).[10] It has been documented that recurrence and progression rates for Tis, Ta, and T1 lesions vary greatly. However, most of the studies reporting on the prognostic value of tumor stage and grade in superficial transitional cell carcinoma have follow-up periods of only 5 years or less.[9-12] Table 2 lists a few of the studies that have followed patients for more than 5 years.[8,13,14] Clinical Studies With Long-Term Follow-up—Heney and colleagues in the National Bladder Cancer Group studied 207 patients with superficial tumors who were followed for a mean of 39 months.[8] Their findings, which are similar to those of other researchers, confirm that higher-grade and higher-stage lesions recur and progress more often. Patients with grade 1, 2, or 3 disease had progression rates to muscle invasive disease of 2%, 11%, and 45%, respectively. In terms of stage, patients with Ta lesions progressed only 4% of the time, as compared with 30% of individuals with T1 tumors. Rates of progression were also higher in patients who had dysplasia elsewhere in the bladder at the time of initial resection. Lamina propria invasion was a significant prognostic factor when combined with grade. Progression was observed in only 6% of patients with grade 2, Ta tumors, as compared with 21% of those with grade 2, T1 lesions. Holmang et al followed 176 patients with primary Ta or T1 tumors for 20 years.[13] All 176 patients were treated initially with transurethral resection (TUR), while 65 patients required either cystectomy or radiation therapy immediately or within a year after TUR. Therefore, the results of the study need to be interpreted with some caution. Overall, the study showed that there were frequent (52%) tumor recurrences, which were confined to the bladder, with a 14% progression rate for Ta lesions and 39% for T1 lesions. The overall risk of dying from superficial bladder cancer was 22% (11% for Ta lesions and 30% for T1 tumors). Similar progression rates were reported by Kurth et al in the European Organization for Research and Treatment of Cancer genitourinary (EORTC-GU) study.[14] Long-term studies have also been performed in “high-risk” populations, including patients with T1 lesions, multiple Ta lesions, or associated Tis. Herr et al reported the 10-year outcome of 221 such patients who had undergone multiple TURs and one or more cycles of intravesical bacillus Calmette-Guérin (BCG) therapy.[15] Tumor progression developed in 42% of patients by 10 years, and superficial tumor recurrences occurred in 49%. Most (82%) of these stage progressions or recurrences had occurred by 5 years. Of note, 18% of patients had a disease-free interval of 5 years or more prior to recurrence or invasion.[1] Proposed Substaging of T1 Lesions—Some researchers have proposed that the current TNM clas-sification system should be modified to include substaging of T1 lesions into T1a and T1b.[16,17] It is felt that this substaging might improve prognostic information. As proposed, T1a Page 2 of 10

Predicting Prognosis in Patients With Superficial Bladder Cance Published on Cancer Network (http://www.cancernetwork.com) lesions would not involve the muscularis mucosa of the lamina propria, whereas T1b lesions may invade into or through this layer. Holmang et al studied 121 patients with T1 transitional cell carcinomas in whom they subclassified 90% of the lesions as either T1a or T1b.[16] The T1b tumors were more often grade 3 than the T1a tumors (79% vs 40%; P < .001). The T1b tumors had a significantly higher overall rate of progression than the T1a lesions (53% vs 31%). The death rate from bladder cancer was nearly twice as high for patients with T1b disease than for those with T1a disease (44% vs 23%; P < .05). Similar results have been reported by Hasui and coworkers.[17] Although this substaging system appears to provide prognostic information using TUR chips, it may be an impractical to use due to technical difficulties, such as the cautery effect, orientation, and lack of observer agreement.[2]

Biological Markers The traditional prognostic factors (cystoscopic findings, tumor stage and grade) are not sufficiently predictive in the individual patient. The results of long-term studies clearly indicate that a better method for predicting superficial bladder cancer behavior is urgently needed. Such a tool may be on the horizon, in the form of a biological marker that accurately predicts which patients are at risk and, therefore, should be treated aggressively from the start. However, for these markers to be employed in clinical practice, they must, in multivariate analysis, yield additional prognostic information beyond that provided by knowledge of tumor grade and stage. DNA Ploidy and S-Phase DNA ploidy, as determined by flow cytometry, has been studied as a potential prognostic marker for bladder cancer progression. Many tumors, including transitional cell carcinoma, have evidence of abnormal amounts of DNA, or aneuploidy. Although studies have indicated that DNA ploidy appears to be related to tumor grade, assessing ploidy has not become a standard of care, and there is no consensus on the usefulness of DNA ploidy as a prognostic marker. For example, the Southwest Oncology Group (SWOG) analyzed 228 patients with superficial transitional cell carcinoma of the bladder to determine whether DNA ploidy is prognostic for disease progression.[18] Nondiploid tumors had higher rates of progression and death than did diploid tumors. However, the information obtained from the DNA analyses was not additive to tumor grade and stage in multivariate analysis.[19] S-Phase Fraction Estimation of tumor proliferation (low vs high S-phase fraction) may be a useful adjunct to DNA ploidy as a potential prognostic marker. S-phase fraction can be estimated from DNA histograms. Automated analysis of such histograms appears to be reliable and can be easily reproduced.[19] In bladder cancer, the proportion of S-phase cells appears to be related to tumor stage. A study by Tribukait showed that the proportion of S-phase cells was increased in T1 tumors compared to Ta tumors in both aneuploid and diploid cancers.[20] A more recent intergroup study (SWOG 8795) was undertaken to assess the prognostic significance of S-phase analysis in stage Ta/T1 bladder cancer.[19] This analysis assessed both recurrence-free and progression-free survival in 223 patients for whom both flow cytometric data and tumor grade were known. One-half of the patients developed bladder cancer recurrence. Of these patients, one-fourth had disease progression. One conclusion that can be drawn from the study is that S-phase fraction (high or low) does provide prognostic information that is additive to tumor grade. Of the patients with high-grade (grade 2 or 3), high–S-phase tumors, 40% had progressed at 24 months following intravesical therapy (BCG or mitomycin). Page 3 of 10

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A series of 194 patients who were followed by Tribukait for > 10 years also showed that S-phase was the single best predictor of disease progression and death.[B. Tribukait, personal communication, 1998] New Technologies—One problem with DNA ploidy and S-phase fraction is that both must be determined by flow cytometry. This technique can be difficult to perform and requires expensive equipment and highly trained personnel. In addition, large sections of tumor are required and, if normal cells are included with the tumor, analysis can be further compromised. In addition, flow cytometry techniques are not standardized. Solutions to this problem may include the use of alternative methods to detect aberrations in cellular DNA content, such as fluorescence in situ hybridization (FISH) and laser scanning cytometry. Fluorescence in situ hybridization allows for visualization and quantitation of chromosomes and genes on a cell-by-cell basis. Technically, this procedure is easy to perform and requires no specialized equipment. Another advantage of FISH over flow cytometry is the former's ability to detect numerical aberrations in specific chromosomes of interest, as opposed to measuring total DNA.[21] Promising data have been published on the ability of FISH to detect DNA aberrations in bladder cancer cells. A study by Sauter and colleagues found a significant difference between Ta and T1 bladder tumors in terms of the fraction of detectable aberrations by FISH (47% vs 85%; P < .0001).[21] This finding supports the concept that Ta and T1 tumors should not be grouped together as “superficial” bladder cancer. Another potential solution to the problem of flow cytometry is the use of laser scanning cytometry. This techniques combines the advantages of flow and image cytometry by using a laser scanning cytometer to measure fluorescence of individual cells rapidly and accurately.[22] Monoclonal Antibodies Several monoclonal antibodies have also been investigated as potential prognostic markers in bladder cancer. Two such antibodies are Ki-67 and proliferating cell nuclear antigen (PCNA), both of which help assess proliferative activity in a variety of neoplasms, including bladder cancer. The rate of cell proliferation is thought to be a significant predictor of tumor behavior and seems to correlate with tumor recurrence, progression, and metastatic potential. Ki-67—The Ki-67 monoclonal antibody has been reported to react with a nuclear antigen that is related to cell proliferation.[23] This antibody therefore labels cells in all phases of the cell cycle except G0. Asakura et al recently investigated whether Ki-67 labeling index can predict recurrence or progression of superficial bladder cancer.[24] Based on an analysis of Ki-67 immunostaining in 104 cases of superficial bladder cancer, these investigators concluded that the recurrence rate was higher in tumors with a labeling index of ≥ 5.35 (P < .001) and correlated with a 70% recurrence rate. By comparison, only 22% of tumors with a labeling index of < 5.35 recurred. Median Ki-67 labeling index was also higher in tumors with progression than in those without (P < .01), although more studies need to be performed before a definite association with progression can be made. However, with increasing importance of S-phase as a prognostic marker, Ki-67 offers a way to assess cell proliferation without employing flow cytometry. PCNA—A study by Chen et al studied a monoclonal antibody to PCNA (PC10) to determine whether PCNA levels are valuable as a prognostic tool in bladder cancer.[25] This antibody is incorporated into the cellular nuclei at the time of DNA synthesis, prior to mitosis. A total of 48 patients were evaluated using a PCNA labeling index. The majority (95.8%) of tumors stained positive for PCNA. The mean labeling index was found to be higher in invasive tumors and in higher-grade tumors. The authors concluded that the PCNA labeling index may be an objective and quantitative assay of tumor aggressiveness and may also provide prognostic information about bladder cancer.[25] Page 4 of 10

Predicting Prognosis in Patients With Superficial Bladder Cance Published on Cancer Network (http://www.cancernetwork.com)

Nuclear Morphometry Other methods of using nuclear markers for predicting the prognosis of bladder cancer have been reported recently. For example, measuring several nuclear morphometric variables has been shown to provide a good indication of prognosis in bladder cancer.[26] These variables include the mean nuclear volume (MNV), nuclear roundness factor (NRF), and variation of nuclear area (VNA). Fukuzawa a colleagues studied these variables in 156 patients and showed that MNV and NRF were prognostic indicators for invasive progression (MNV, P = .02; NRF, P = .009).[26] Cell-Cycle Regulators Investigators also have been studying mutations in various cell-cycle regulators and the association of these mutations with bladder tumor behavior. There are many complex interactions among the known cell-cycle regulators, and sophisticated studies have been undertaken to help delineate which of these interactions and mutations are important in disease progression. Examples of some of the more important “players” in cancer are several cyclin-dependent kinase (CDK) inhibitors, p15, p16, and p21, and two tumor-suppressor genes, p53 (alias TP53) and Rb. Figure 1 depicts the relationship between these genes and the cell cycle. CDK Inhibitors—The cyclins and CDKs constitute the protein complexes that control cellular proliferation.[27] Cyclins can function as regulatory molecules through phosphorylation of certain elements, such as the Rb protein. Many CDKs have been discovered that can form complexes with cyclins and serve as catalytic subunits. An example is CDK2, which complexes with cyclin E and controls entry into S-phase.[27] Recently, negative cell-cycle regulators have been discovered that inhibit CDK catalytic subunits by forming stable complexes with the CDKs. Examples are p21 and p16, the latter of which inhibits the activity of CDK4 and CDK6. This, in turn, inhibits Rb phosphorylation. The p15/INK4B gene is similar to p16 and appears to inactivate many of the same CDKs.[27] Mutations of these cell-cycle regulators are being discovered and analyzed as potential markers for high-risk bladder tumors. p53—The p53 tumor-suppressor gene has been mapped to chromosome 17 and encodes for a 53-kd nuclear phosphoprotein. Wild-type p53 is a checkpoint regulator that mediates apoptosis or cell-cycle arrest in G1 in response to DNA damage.[28] This cell-cycle arrest allows the cells to repair their DNA. Mutations of the p53 gene can lead to unregulated cell proliferation, tumor progression, and insensitivity to chemotherapy or radiotherapy.[29] Mutations of p53 are the most common molecular abnormalities found in human tumors. The mutations occur in a variety of malignancies including bladder cancer. It has been suggested that different mutations of p53 may invoke different biological behavior of the tumors. Deletions, point mutations, or interactions with other proteins can cause inactivation or mutations of TP53. Mutations can be detected by single-strand conformation polymorphism (SSCP) or by direct sequencing.[30] Early studies comparing p53 nuclear protein accumulation by immunohistochemistry and mutations in the p53 gene also found a possible relationship between positive staining and p53 gene mutations.[31,32] These studies failed to show an absolute association between the presence of p53 mutations and immunohistochemical overexpression, however. A study of 73 bladder tumors also correlated p53 alterations (using immunohistochemistry and molecular methods) to higher grade (P = .003) and stage (P = .01) of tumors—factors that are known to correlate with bladder cancer progression.[31] Evidence suggests that the p53 gene plays a significant role in cancer progression, but we still do not know whether p53 protein overexpression or alterations can be used as a reliable prognostic marker. Several studies have retrospectively reviewed the power of p53 overexpression (by immunohistochemistry) and/or p53 abnormalities to predict tumor progression and patient survival.[33-38] Table 3 lists some of the studies that evaluate nuclear overexpression of p53 as a marker for disease progression. In general, these studies show that p53 overexpression correlates Page 5 of 10

Predicting Prognosis in Patients With Superficial Bladder Cance Published on Cancer Network (http://www.cancernetwork.com) with a higher probability of disease progression. A study by Thomas and colleagues demonstrated that all tumors with abnormal amounts of DNA and all high-grade tumors overexpressed p53.[34] Patients with high levels of p53 staining (> 10% nuclear staining) progressed more often than those with low levels (P = .007). Nevertheless, the same study showed that tumor grade was a more specific predictor of progression, since all grade 3 tumors progressed. A similar study by Serth and coworkers involved 69 patients with stage T1 bladder cancer.[38] Patients with p53-positive tumors (> 20% of cells staining positive) had a much higher rate of progression than did those with negatively staining tumors (86% vs 2%). Although these reports provide support for the p53 gene as a potential prognostic marker, it is important to note that almost all of the studies used different techniques.[39] Most researchers employ immunohistochemical techniques, but the antibodies used often differ, and the criteria for positivity vary widely. These differences do not allow the technique to be universal or easily applied by the clinician. Therefore, further studies are needed to confirm the current results and attempt to provide standardization. Large, prospective studies will also be helpful in determining whether p53 overexpression or mutation truly correlates with treatment outcome. Rb Gene—It has been determined that the Rb tumor-suppressor gene not only is related to the development of retinoblastoma but also plays a key role in the development and/or progression of many common malignancies, including bladder cancer. The RB gene encodes a 110-kd nuclear phosphoprotein (pRb), which is believed to function as a cell-cycle regulator.[40] The functionally active form of Rb is thought to be the hypophosporylated form, which exerts a negative regulatory effect on gene expression through formation of a complex with DNA-binding proteins.[41] Cyclin-dependent kinase complexes can hyperphosphorylate Rb and render it inactive. This allows cells to pass from G1 to the S-phase of the cell cycle and, therefore, makes Rb an important cell-cycle regulator.[42] It has been suggested that alterations in Rb, specifically, loss of Rb expression, may correlate with higher tumor grades and stages and, therefore, with prognosis in bladder cancer.[43,44] A study by Cordon-Cardo and coworkers showed that patients with muscle invasive tumors had altered Rb expression more often than those with superficial tumors. In addition, 5-year survival was significantly lower among those patients with altered Rb protein levels, as compared with patients with normal levels of Rb expression (P < .001).[45] Like p53, Rb has not been studied thoroughly enough to make it a good prognostic marker. Encouraging data suggest that Rb may play a significant role in bladder cancer pathogenesis, but it may be more closely associated with higher-stage disease. However, one interesting study has examined the cooperative effects of p53 and pRb alterations in superficial bladder cancer. This study showed that patients whose tumors showed both p53 overexpression and altered Rb expression (undetectable levels) had a marked increase in dis-ease progression and reduced overall survival.[46] Other Biological Markers Other potentially important prognostic markers include cell adhesion molecules (E-cadherin), the oncogene c-erb-B2, and components of the apoptosis pathways (bcl-2, bax, and bcl-X).[47,48] Also, many other potential prognostic factors for bladder cancer are still in the early stages of investigation. For example, tumor angiogenesis, as determined by measurement of microvesical density, has been associated with both disease-free and overall survival in bladder cancer patients. Patients with the lowest microvessel counts were found to have a 68% 5-year survival rate, as compared with a 34% rate for those with the highest microvessel counts.[49] In addition, angiogenesis was associated with tumor progression and was found to be an independent prognostic indicator after adjustment for Page 6 of 10

Predicting Prognosis in Patients With Superficial Bladder Cance Published on Cancer Network (http://www.cancernetwork.com) tumor grade, stage, and regional lymph node status.

Conclusions At present, clinicians can assure patients with Ta bladder tumors that they have a very low likelihood of progressing to muscle invasive disease. Regrettably, for patients with T1, grade 2 or 3 lesions, little can be done currently to predict, at presentation, who will progress. At present, the most promising prognostic markers appear to be S-phase, p53, and Rb status. Hopefully, the advent of new technology, such as laser scanning cytometry, will allow all of these markers to be assessed simultaneously on a single biopsy specimen. This may provide the knowledge needed to predict which patients with superficial bladder cancer should be treated aggressively from the outset.

References: 1. American Cancer Society: Cancer Facts and Figures 1998, pp 1-18. Atlanta, Georgia, American Cancer Society, 1998. 2. Ismail MT, Lattime EC, Gomela LG: Current management of superficial bladder cancer: BCG and beyond. Monogr Urol 19:33-55, 1998. 3. Clayson DA, Cooper EH: Cancer of the urinary tract. Adv Cancer Res 13:271-381, 1970. 4. Price JM: Etiology of Bladder Cancer: Benign and Malignant Tumors of the Urinary Bladder, pp 189-251. Flushing, New York, Medical Examination Publishing, 1971. 5. Ross RK, Paganini-Hill A, Henderson B: Diagnosis and Management of Genitourinary Cancer, pp 23-31. Philadelphia, Saunders, 1988. 6. Herr HW: Natural history of superficial bladder tumors: 10-20 year follow-up of treated patients. World J Urol 15:84-88, 1997. 7. Heney NM: Natural history of superficial bladder cancer: Prognostic features and long-term disease course. Urol Clin North Am 19:429-433, 1992. 8. Heney NM, Ahmed S, Flanagan MJ: Superficial bladder cancer: Progression and recurrence. J Urol 130:1083-1086, 1983. 9. Fitzpatrick JM, West AB, Butler MR: Superficial bladder tumors: The importance of recurrence pattern following initial resection. J Urol 135:920-924, 1986. 10. Sobin LH, Wittekind CH (eds): TNM Classification of Malignant Neoplasms. New York, Wiley-Liss, 1997. 11. Lutzeyer W, Rubben H, Dahm H: Prognostic parameters in superficial bladder cancer: An analysis of 315 cases. J Urol 127:250-252, 1982. 12. Torti FM, Lum BL, Aston D, et al: Superficial bladder cancer: The primacy of grade in the development of invasive disease. J Clin Oncol 5:125-130, 1987. 13. Holmang S, Hedelin H, Anderstrom C, et al: The relationship among multiple recurrences, progression ad prognosis of patients with stages Ta and T1 transitional cell cancer of the bladder followed for at least 20 years. J Urol 153:1823-1826, 1995. 14. Kurth K, Tunn U, Ay R, et al: Adjuvant chemotherapy for superficial transitional cell bladder carcinoma: Long-term results of a European Organization for Research and Treatment of Cancer Page 7 of 10

Predicting Prognosis in Patients With Superficial Bladder Cance Published on Cancer Network (http://www.cancernetwork.com) randomized trial comparing doxorubicin, ethoglucid and transurethral resection alone. J Urol 158:378-384, 1997. 15. Herr HW, Wartinger DD, Fair WR, et al: Bacillus Calmette-Guerin therapy for superficial bladder cancer: A 10-year follow-up. J Urol 147:1020-1023, 1992. 16. Holmang S, Hedelin H, Anderstrom C, et al: The importance of the depth of invasion in stage T1 bladder carcinoma: A prospective cohort study. J Urol 157:800-804, 1997. 17. Hasui Y, Osada Y, Kitada S, et al: Significance of invasion to the muscularis mucosa on the progression of superficial bladder cancer. Urology 43:782-786, 1994. 18. deVere White RW, Deitch AD, Tesluk H, et al: Prognostic significance of DNA ploidy in Ta/T1 bladder cancer: A Southwest Oncology Group study. Urol Oncol 2:27-34, 1996. 19. deVere White RW, Deitch AD, Daneshamand S, et al: The prognostic signficance of S-phase analysis in stage Ta/T1 bladder cancer: A Southwest Oncology Group study (abstract 552). Presented at the 1998 American Urological Association meeting. 20. Tribukait B: Tumor biology in diagnostic cytology: DNA cytometry in carcinomas of the bladder and prostate. Recent Results Cancer Res 133:23-31, 1993. 21. Sauter G, Gasser TC, Moch H, et al: DNA aberrations in urinary bladder cancer detected by flow cytometry and FISH. Urol Res 25(suppl 1):S37-S43, 1997. 22. Kamentsky LA, Kamentsky LD: Microscope-based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data. Cytometry 12:381-387, 1991. 23. Stavropoulos NE, Ioackim-Velogianni E, Hastazeris K, et al: Growth fractions in bladder cancer defined by Ki-67: Association with cancer grade, category and recurrence rate of superficial lesions. Br J Urol 72:736-739, 1993. 24. Asakura T, Takano Y, Iki M, et al: Prognostic value of Ki-67 for recurrence and progression of superficial bladder cancer. J Urol 158:385-388, 1997. 25. Chen G, Lin MS, Li RC: Expression and prognostic value of proliferating cell nuclear antigen in transitional cell carcinoma of the urinary bladder. Urol Res 25:25-30, 1997. 26. Fukazawa S, Hashimura T, Sasaki M, et al: Nuclear morphometry for improved prediction of the prognosis of human bladder carcinoma. Cancer 76:1790-1796, 1995. 27. Dalbagni G, Cordon-Cardo C: Mutations of the cell-cycle regulators in bladder carcinoma. Mol Urol 1:39-44, 1997. 28. Kastan M, Onkyekwere O, Sidransky D, et al: Participation of p53 protein in response to DNA damage. Cancer Res 51:6304-6311, 1991. 29. Velculescu VE, El-Demy WS: Biologic and clinical importance of the p53 tumor suppressor gene. Clin Chem 42:858-868, 1996. 30. Hollstein M, Sidransky D, Vogelstein B, et al: p53 Mutations in human cancers. Science 253:49-53, 1991. 31. Esrig D, Spruk III CH, Nichols PW, et al: p53 Nuclear protein accumulation correlates with mutations in the p53 gene, tumor grade, and stage in bladder cancer. Am J Pathol 143:1389-1397, 1993. 32. Cordon-Cardo C, Dalbagni G, Saez GT, et al: p53 Mutations in human bladder cancer: Genotypic vs phenotypic patterns. Int J Cancer 56:347-353, 1994. Page 8 of 10

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33. Sarkis AS, Dalbagni G, Cordon-Cardo C, et al: Nuclear overexpression of p53 protein in transitional cell bladder carcinomas: A marker of disease progression. J Natl Cancer Inst 85:53-59, 1993. 34. Thomas DJ, Robinson MC, Charlton R, et al: p53 Expression ploidy and progression in pT1 transitional cell carcinoma of the bladder. Br J Urol 73:533-537, 1993. 35. Lacombe L Dalbagni G, Zhang Z-F, et al: Overexpression of p53 protein in a high-risk population of patients with superficial bladder cancer before and after bacillus Calmette-Guerin therapy: Correlation to clinical outcome. J Clin Oncol 14:2646-2652, 1996. 36. Vet JAM, Witues A, Marras SAE, et al: Predictive value of p53 mutations analyzed in bladder washings for progression of high-risk superficial bladder cancer. Clin Cancer Res 2:1055-1061, 1996. 37. Liukkonen TJO, Lipponen PD, Helle M, et al: Immunoreactivity of bcl-2, p53 and EGFr is associated with tumor stage, grade, and cell proliferation in superficial bladder cancer. Urol Res 25;1-8, 1997. 38. Serth J, Kuczk MA, Bokemeyer C, et al: p53 Immunohistochemistry as an independent prognostic factor for superficial transitional cell carcinoma of the bladder. Br J Cancer 71:201-205, 1995. 39. Malkowicz SB: Superficial bladder cancer: The role of molecular markers in the treatment of high-risk superficial disease. Semin Urol Oncol 15:169-178, 1997. 40. Goodrich DW, Wang NP, Quian Y-W, et al: The retinoblastoma gene product regulates progression throughout the G1 phase of the cell cycle. Cell 67:293-302, 1991 41. Mittnacht S, Weinberg RA: G1/S phosphorylation of the retinoblastoma protein is associated with an altered affinity for the nuclear compartment. Cell 65:381-393, 1991. 42. Riley DJ, Lee YH, Lee WH: The retinoblastoma protein: More than a tumor suppressor. Annu Rev Cell Biol 10:1-29, 1994. 43. Presti JC Jr, Reuter VE, Galan T, et al: Molecular genetic alterations in superficial and locally advanced human bladder cancer. Cancer Res 51:5404-5409, 1991. 44. Xu H-J, Cairns P, Hu S-X, et al: Loss of RB protein expression in primary bladder cancer correlates with loss of heterozygosity at the RB locus and tumor progression. Int J Cancer 53:781-784, 1993. 45. Cordon-Cardo C, Wartinger D, Petrylak D, et al: Altered expression of the retinoblastoma gene product: Prognostic indicator in bladder cancer. J Natl Cancer Inst 84:1251-1256, 1992. 46. Cordon-Cardo C, Zhang ZF, Dalbagni G, et al: Cooperative effects of p53 and pRB alterations in primary superficial bladder tumors. Cancer Res 57:1217-1221, 1997. 47. Lipponeu PK, Eskelinen MJ: Reduced expression of E-cadherin is related to invasive disease and frequent recurrence in bladder cancer. J Cancer Res Clin Oncol 121:303-308, 1995. 48. Kirsh EJ, Baunoch DA, Stadler WM: Expression of bcl-2 and bcl-x in bladder cancer.J Urol 159:1348-1353, 1998. 49. Bochner BH, Cote RJ, Weidner N, et al: Angiogenesis in bladder cancer: Relationship between microvessel density and tumor progression. J Natl Cancer Inst 87:1603-1612, 1995.

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