Pretreatment Proliferation and Local Control in Bladder Cancer after Radiotherapy with or without Concurrent Chemotherapy

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1 Strahlentherapie und Onkologie Original Article Pretreatment Proliferation and Local Control in Bladder Cancer after Radiotherapy with or without Concurrent Chemotherapy Christian Weiss 1, 2, Franz Rödel 1, 2, Oliver Ott 1, Dirk Gerhard Engehausen 3, Thomas Papadopoulos 4, Rolf Sauer 1, Claus Rödel 1, 2 Purpose: To investigate whether the addition of chemotherapy to radiotherapy (RT) is beneficial particularly in bladder tumors that possess the capacity for rapid proliferation. Patients and Methods: The Ki-67 index was evaluated by immunohistochemistry on pretreatment biopsies from 136 patients treated by transurethral tumor resection (TURBT) and RT (n = 50) or platin-based radiochemotherapy (RCT; n = 86). Ki-67 expression was correlated with response to RT/RCT and long-term local control rates. The median follow-up was 43 months. Results: The percentage of Ki-67-positive cells ranged from 1.5% to 89%. Complete response (CR) was observed in 100/131 patients (76%, five without restaging TURBT). A statistically significant association between high Ki-67 index ( median) and CR was noted for patients receiving RCT (93% vs. 66% for Ki-67 < median; p = 0.001), but not for patients treated with RT alone (p = 0.12). Long-term local control was 39% for patients treated with RT, and 44% for patients after RCT (p = 0.49). Patients with high Ki-67 index did significantly better when subjected to combined RCT (55% vs. 33% with low Ki-67 index; p = 0.006), whereas no difference between high and low Ki-67 status was observed in the RT group (39% each; p = 0.57). On multivariate analysis, Ki-67 status was an independent predictor for local failure in the RCT group (risk ratio, 0.43; p = 0.007). Disease-specific survival was significantly better after RCT (62%) as compared with RT (42%; p = 0.03), however, the Ki-67 index was not related to this endpoint. Conclusion: Rapid proliferation is associated with improved local control, if patients are treated with concurrent RCT. The cytostatic effect of concurrent chemotherapy may effectively inhibit repopulation during fractionated RT. Key Words: Bladder cancer Radiochemotherapy Ki-67 Local control Strahlenther Onkol 2007;183:552 6 DOI /s Prätherapeutische Proliferationsrate und lokale Kontrolle beim Harnblasenkarzinom nach Radiotherapie mit oder ohne simultane Chemotherapie Ziel: Untersuchung der Frage, ob eine simultane Radiochemotherapie (RCT) besonders bei Harnblasenkarzinomen mit hoher Proliferationsrate zu günstigen Ergebnissen führt. Patienten und Methodik: Der Ki-67-Index wurde immunhistochemisch an prätherapeutischen Biopsien von 136 Patienten untersucht, die nach transurethraler Tumorresektion (TURBT) mittels Bestrahlung (RT; n = 50) oder cisplatinbasierter RCT (n = 86) behandelt wurden. Die Ki-67-Expression wurde mit dem Tumoransprechen nach RT/RCT und mit der lokalen Kontrollrate korreliert. Die mediane Nachbeobachtungszeit betrug 43 Monate. Ergebnisse: Der Prozentsatz Ki-67-positiver Zellen lag zwischen 1,5% und 89%. Eine Vollremission (CR) wurde bei 100/131 Patienten erreicht (76%, fünf Patienten erhielten keine Restaging-TURBT). Eine statistisch signifikante Assoziation zwischen hohem Ki-67-Index ( Median) und CR bestand für Patienten, die eine RCT erhielten (93% vs. 66% für Ki-67 < Median; p = 0,001), aber nicht für Patienten mit alleiniger RT (p = 0,12). Eine langfristige lokale Kontrolle konnte bei 39% der mit RT und bei 44% der mit RCT behandelten Patienten erreicht werden (p = 0,49). Bei Patienten mit hohem Ki-67 war die lokale Kontrolle signifikant besser, wenn sie eine RCT erhielten (55% vs. 33% mit niedrigem Ki-67-Index; p = 0,006), wohingegen in der Gruppe der ausschließlich bestrahlten Patienten kein Unterschied in der lokalen Kontrolle zwischen Tumoren mit hohem und niedrigem Ki-67-Status beobachtet wurde (39% für beide Gruppen; p = 0,57). In der multivariaten Analysis war der Ki-67-Status ein unabhängiger Prädiktor 1 Department of Radiation Therapy, University of Erlangen, Germany, 2 Department of Radiation Therapy, University of Frankfurt/Main, Germany 3 Department of Urology, University of Erlangen, Germany, 4 Institute of Pathology, University of Erlangen, Germany. Received: April 30, 2007; accepted: August 7, Strahlenther Onkol 2007 No. 10 Urban & Vogel

2 für die lokale Kontrolle in der RCT-Gruppe (relatives Risiko 0,43; p = 0,007). Das krankheitsspezifische Überleben war nach RCT (62%) signifikant besser als nach RT (42%; p = 0,03). Der Ki-67-Index war mit diesem Endpunkt nicht assoziiert. Schlussfolgerung: Eine hohe Proliferationsrate ist mit einer verbesserten lokalen Kontrolle verbunden, wenn Patienten eine simultane RCT erhalten. Der zytostatische Effekt der simultanen Chemotherapie wirkt möglicherweise der Tumorzellrepopulierung während fraktionierter RT entgegen. Schlüsselwörter: Blasenkarzinom Radiochemotherapie Ki-67 Lokale Kontrolle Introduction Proliferation characteristics are likely to play a key role in the response of tumors to radiation [6, 18]. Repopulation of tumor cells during fractionated radiotherapy (RT) is one of the mechanisms by which tumors may evade the cytotoxic effect of RT, a process that may even be accelerated during the course of fractionated RT [2]. In bladder cancer, a retrospective analysis by Maciejewski & Majewski suggested that tumor clonogenic repopulation accelerates after a lag period of 5 weeks and that a dose increment of 0.36 Gy per day is required to compensate for this repopulation [13]. Attempts to overcome this mechanism of resistance have concentrated on altering dose-time-fractionation pattern, particularly by adopting accelerated schedules. Horwich et al. reported the results of a randomized study in 229 patients that either received 60.8 Gy in 32 fractions over 26 days or 64 Gy in 32 fractions over 45 days. The accelerated schedule did not improve local control or survival and was associated with increased complications [10]. Another strategy is to target tumor cell repopulation by the addition of chemotherapy an approach that has extensively been studied in the RTOG series and in European protocols [1, 5, 17, 21, 24, 25]. The only randomized comparison of RT versus radiochemotherapy (RCT) in bladder cancer showed an improved local control when cisplatin was given with RT [4]. The goal of this study was to investigate the hypothesis that concurrent RCT is particularly beneficial in patients whose tumors possess the capacity for rapid proliferation. For this purpose, we have retrospectively analyzed the Ki-67 index in a cohort of 136 patients, 50 of whom were treated with fractionated RT, and 86 received platin-based concomitant RCT. Ki-67 is a nuclear protein expressed by proliferating cells and can be observed immunohistochemically [7, 15]. Patients and Methods Patient and Tumor Characteristics Between 1982 and 2000, a total of 415 patients with bladder cancer were treated. During this 18-year period, patients initially received RT alone ( ); since 1985 platin-based chemotherapy has been administered concurrently with RT [21]. Having been compiled for research purposes, the group of 136 patients investigated for this analysis represents those for whom pretreatment archival tissue blocks, clinical followup and data on local control were available. No further selection criteria were applied. 50 patients were treated with RT alone, 126 received platin-based RCT. The T-category, grading, and other clinicopathologic factors are shown in Table 1. Treatment Protocol Treatment was commenced by transurethral tumor resection (TURBT) aimed at maximal reduction of the tumor. RT was initiated 4 8 weeks after TURBT, using 6- to 15-MV photons and a four-field box technique with individually shaped portals and daily fractions of 1.8 Gy on 5 days per week. Patients received Gy to the pelvic lymph nodes, then a cone-down was applied to boost the bladder to 55.4 Gy after complete resection, and to 59.4 Gy after incomplete resection. Cisplatin was given before each RT fraction on days 1 5 and at a dose of 25 mg/m 2 /d, or at a dose of 20 mg/m 2 /d when 5-fluorouracil (600 mg/m 2 /d, 120-h infusion) was applied as a second agent. 6 weeks after RT/RCT, response was evaluated by TURBT of the former tumor bed. In case of complete response (CR), patients were followed at 3-month intervals, including cystoscopy. In case of residual tumor at restaging TURBT or invasive local failure, salvage cystectomy was recommended [21]. Immunohistochemical Staining of Ki-67 Formalin-fixed, paraffin-embedded tissue sections were deparaffinized as previously described [23]. To undo masking effects of formalin fixation, sections were overlain with 10 mm citrate buffer (ph 6.0) and heated for 15 min in a microwave oven at 750 W. When the slides and buffer were at room temperature, the slides were rinsed in phosphate-buffered saline (PBS); nonspecific binding sites were blocked with 5% bovine serum albumin in PBS. After washing in PBS, the monoclonal mouse antibody MIB-1, that reacts to a nuclear antigen Ki-67 (Dianova, Hamburg, Germany, dilution 1 : 30) was applied and incubated for 2 h at room temperature. Negative controls were created by omitting the primary antibody. Tumor sections that showed intense positivity for Ki-67 were used as positive controls. The samples were incubated with biotinylated rabbit anti-mouse secondary antibody (Dianova, dilution 1 : 50, incubated for 1 h at room temperature) followed by an avidin/biotylinated alkaline phosphatase and Fast-Red solution. The Ki-67 index, defined as the percentage of positive carcinoma cells, was determined after evaluation of at least 1,000 carcinoma cells for Ki-67 immunoreactivity using the previousy described Image System [23]. Strahlenther Onkol 2007 No. 10 Urban & Vogel 553

3 Table 1. Association of Ki-67 immunostaining with pretreatment tumor characteristics in 136 patients with bladder cancer. SD: standard deviation; TURBT: transurethral resection of bladder tumor. Tabelle 1. Assoziation zwischen Ki-67-Immunfärbung und prätherapeutischen Tumorcharakteristika bei 136 Patienten mit Harnblasenkarzinom. SD: Standardabweichung; TURBT: transurethrale Resektion des Blasentumors. Patients (n) p-value Ki-67 Index Mean (± SD) All patients (17.8) Gender Male (18.3) 0.79 Female (15.8) Age < Median (18.7) 0.93 Median (17.3) T-category T (15.4) 0.74 T (17.5) T (21.2) Resection status after initial TURBT R (15.5) 0.40 R (18.6) R (17.5) Tumor grading G (8.3) 0.08 G (16.4) G (19.0) Associated carcinoma in situ Yes (16.7) 0.47 No (18.6) Unknown 13 Multifocality Yes (18.7) 0.47 No (17.9) Unknown 12 Statistical Analysis The main endpoint was to investigate the role of Ki-67 index as predictor for response at restaging TURBT and long-term local control, because this endpoint is directly related to the local treatment efficacy of RT and RCT, and stratifies patients for bladder preservation or salvage cystectomy. For local control rates any local failures to RT/RCT, i.e., persistent tumor at restaging TURBT as well as any superficial or muscle-invasive relapse after initial CR during follow-up, were counted as events. Secondary endpoints included metastases-free survival and disease-specific survival. Survival rates were calculated from time of diagnosis to the occurrence of distant metastases or last follow-up for metastases-free survival, and death from bladder cancer or last follow-up for disease-specific survival. To obtain predictive values, the Ki-67 index was subdivided into two groups with the median as cutpoint. Continuous variables were compared between the groups using the t-test for independent samples and, in case of more than two groups, the one-way ANOVA. Categorial data were compared using the χ 2 -test, censored data using the log-rank test. Multivariate analyses were done using the Cox proportional hazard regression model. The level of significance was 0.05 in all statistical testing. Results Immunostaining Ki-67 Study and Pretreatment Clinical Factors The percentage of Ki-67-positive cells in 136 biopsies ranged from 1.5% to 89%, with a mean of 20.5% and a median of 16.7%. The mean proliferation index for G1, G2, and G3 tumors was 6.5% (n = 5 patients), 18.9% (n = 60 patients), and 23.1% (n = 71 patients), respectively (p = 0.08; Table 1). No significant association was found between the Ki-67 index and other clinicopathologic factors (Table 1). Response at Restaging TURBT and Long-Term Local Control CR at restaging TURBT was observed in 100/131 patients (76%, five patients without restaging TURBT). CR was 68% after RT alone, and 81% after RCT (p = 0.14). As shown in Table 2, a statistically significant association between Ki-67 index and CR was noted for patients receiving RCT, but not for patients treated with RT. The median follow-up time for all patients was 43 months (range: months). 5-year local control without any superficial or muscle-invasive relapse was 42% for the entire cohort of 136 patients; it was 39% for patients treated with RT alone, and 44% for patients after RCT (p = 0.49). Patients with high Ki-67 index ( median) did significantly better when treated by combined RCT (p = 0.006), whereas no difference between high and low Ki-67 was observed in the RT group (Figures 1a and 1b). This remains also true, if not the median is taken as cutoff, but the 33.33% percentile (low/intermediate/high Ki-67 index): local control for the RCT group was then Table 2. Initial response at restaging TURBT according to Ki-67 index and treatment (RT alone/rct). CR: complete response; RCT: radiochemotherapy; RT: radiotherapy; TURBT: transurethral resection of bladder tumor. Tabelle 2. Initiales Tumoransprechen bei der Restaging-TURBT gemäß Ki-67-Index und Therapieart (RT allein/rct). CR: Vollremission; RCT: Radiochemotherapie; RT: Radiotherapie; TURBT: transurethrale Resektion des Blasentumors. CR at restaging No CR at p-value TURBT restaging TURBT n (%) n (%) RT alone a Ki-67 < median (n = 27) 16 (59) 11 (41) 0.12 Ki-67 median (n = 20) 16 (80) 4 (20) RCT a Ki-67 < median (n = 38) 25 (66) 13 (34) Ki-67 median (n = 46) 43 (93) 3 (7) a 3 and 2 patients without restaging TURBT in RT and RCT group, respectively 554 Strahlenther Onkol 2007 No. 10 Urban & Vogel

4 Local control a 1,0 0,8 0,6 0,4 0,2 0,0 RT alone (n = 50) 1,0 Platin-based Ki-67 median 0,8 Ki-67 median RCT (n = 86) 0,6 55% 39% 0,4 33% Ki-67 < median Ki-67 < median 0,2 p = ,0 p = Time (months) b Time (months) Figures 1a and 1b. Local control according to treatment (RT [a], and RCT [b]) and Ki-67 proliferation index in patients treated for invasive bladder cancer. Abbildungen 1a und 1b. Lokale Kontrolle nach Therapieart (RT [a] und RCT [b]) und Ki-67-Index bei Patienten mit invasivem Harnblasenkarzinom. 28%/44%/63% (p = 0.01), and for the RT group 47%/16%/ 55% (p = 0.51). On multivariate analysis, the Ki-67 status was the only independent factor for local control after adjusting for other parameters in the RCT group (Table 3). Survival Data Metastases-free survival for the entire group was 69% at 5 years; it was 76% and 68% for patients treated with RT and RCT (p = 0.52), respectively. The Ki-67 index was not related to the risk of developing distant metastases. This was true for all patients (5-year metastases-free survival: low Ki-67: 69%, and high Ki-76: 71%; p = 0.84) as well as for patients treated with RT (82% vs. 67%; p = 0.83) and RCT (64% vs. 70%; p = 0.62). Disease-specific survival at 5 years was 56% for all patients; it was significantly better for patients treated with RCT Table 3. Multivariate Cox regression analysis of Ki-67 status and pathologic features for the prediction of local failure in 86 patients with invasive bladder cancer treated with transurethral tumor resection (TURBT) and radiochemotherapy. CI: confidence interval. Tabelle 3. Multivariate Cox-Regression des Ki-67-Status und histopathologischer Faktoren bezüglich der lokalen Kontrolle bei 86 Patienten mit Harnblasenkarzinom, die nach transurethraler Tumorresektion (TURBT) eine Radiochemotherapie erhielten. CI: Konfidenzintervall. Local control Local failure Risk ratio (95% CI) p-value Ki-67 < median median 0.43 ( ) Resection status after initial TURBT R0 R ( ) 0.82 R ( ) 0.15 T-category T1 T ( ) 0.33 T ( ) 0.28 Tumor grading G1 2 G ( ) 0.09 (62%) as compared with RT (42%; p = 0.03). The Ki-67 index was not related to this endpoint for all patients, and not for the RT and RCT subgroups. Discussion The fraction of proliferating cells as determined by Ki-67 is considered to be an adverse prognostic feature in bladder cancer. A significant correlation between increased expression of Ki-67 and unfavorable clinicopathologic characteristics, such as grade and stage, has been demonstrated [14, 19]. More recently, molecular studies have correlated high Ki-67 expression with elevated levels of the epidermal growth factor receptor (EGFR), and with higher expression of survivin both negative prognostic factors for many malignancies [20, 27]. Not surprisingly, significantly higher recurrence rates have been reported in surgically managed bladder tumors with a high proliferative index [14, 20]. The role of Ki-67 in predicting local control after RT in bladder cancer is less clear [26]. In a series of 83 patients treated with RT alone, Moonen et al. could not establish a significant correlation between the pretreatment Ki-67 index and local control rates [16]. Likewise, in a series of 55 patients treated with RT alone, Lara et al. did not find the Ki-67 index to be related with local control rates when the median was selected as the cutoff value [12]. This is in line with our findings in 50 patients treated with RT alone. The discrepancy between surgical and RT series with respect to the prognostic impact of tumor cell proliferation remains to be clarified. One possible explanation may arise from the radiobiological as well as clinical experience, whereby rapidly proliferating tumors are more sensitive to RT than those growing slowly. Moreover, higher levels of Ki-67 have been detected in well-oxygenated, more radioresponsive, as opposed to hypoxic, more radioresistant, bladder tumors [11]. Overall, the favorable (more cells in radiosensitive cell-cycle phases, well-oxygenated tumor) and unfavorable features (regrowth between fractions, linkage with EGFR and survivin expression) in rapidly proliferating tumors may thus balance each other when patients are treated with RT. This is consistent with the clinical observation, that the pretreatment Ki-67 index is neither a positive nor a negative predictor for local control in patients treated with RT alone. By contrast, we found that increased pretreatment proliferation is an independent predictor for tumor response and local control when chemotherapy was given in conjunction with RT. Chemotherapy, especially cisplatin, exerts its cytotoxic as well as radiosensitizing potential when cells proliferate into the S/G2/M phases. This effect may be less operative, and, consequently, RCT less effective, when cells are arrested in the G0 phase, as indicated by a low Ki-67 index. Moreover, Strahlenther Onkol 2007 No. 10 Urban & Vogel 555

5 the cytostatic effect of chemotherapy may effectively inhibit repopulation during the course of fractionated RT. Overall, the net effects of proliferation would then favor to apply RCT especially in tumors with a high pretreatment Ki-67 index. This has also been shown in other tumor types, such as head-and-neck, anal and rectal cancer treated by definitive RCT [8, 9, 22]. We are not aware of any other group that has investigated the relationship between Ki-67 and treatment outcomes after combined RCT within bladder-sparing treatment programs. The RTOG Genitourinary Translational Research Group has recently published results from 73 patients treated within the RTOG bladder cancer trials (all with concurrent RCT) [3]. This group did not specifically address the issue of tumor cell proliferation. However, they found that EGFR expression was intriguingly associated with improved response to RCT a finding that would not have been predicted from other tumor types. A strong association between EGFR expression and cell proliferation has been shown in bladder cancer. Thus, in the light of the data presented here, it can be hypothesized that EGFR may be a molecular marker of enhanced proliferation which in turn renders cells more responsive to RCT. Conclusion The results of this study and data from the literature would support a scenario in which high Ki-67 expression is associated with an aggressive phenotype and an unfavorable prognosis when treated by surgery alone, yet these effects may be counteracted and balanced in part when treated with RT. With the addition of chemotherapy to RT, rapid proliferation may render cells particularly responsive to the effects of chemotherapy, RT, and their interactions. References 1. Baierlein SA, Distel L, Sieber R, et al. Combined effect of tumor necrosis factor-alpha and ionizing radiation on the induction of apoptosis in 5637 bladder carcinoma cells. Strahlenther Onkol 2006;182: Bentzen SM. 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Strahlenther Onkol 2005;181: Wu Y, Wang G, Wei J, et al. Survivin protein expression positively correlated with proliferative activity of cancer cells in bladder cancer. Indian J Med Sci 2005;59: Address for Correspondence Prof. Dr. Claus Rödel Department of Radiation Therapy University of Frankfurt Theodor-Stern-Kai Frankfurt/Main Germany Phone (+49/69) , Fax claus.roedel@kgu.de 556 Strahlenther Onkol 2007 No. 10 Urban & Vogel