Immunohistochemical analysis of Ki-67, p21 waf1/cip1 and apoptosis in marker lesions from patients with super®cial bladder tumours treated with vinorelbine intravesical therapy in a preliminary phase I trial R.D. BONFIL, A.D. GONZALEZ, D. SIGUELBOIM, F.D. CUELLO CARRION, D.R. CIOCCA, A. VILLARONGA, L. METZ, F. MOSSO, E. FAYAD, M. REALE and A.J. SCHMILOVICH Laboratory of Fundacio Ân de Investigacio Ân del Ca Âncer at CEFYBO, Buenos Aires, Laboratory of Reproduction and Lactation at CRICYT, Mendoza, Hospital Me Âdico Policial Churruca (Visca), Buenos Aires, Institutos Me Âdicos Anta Ârtida, Buenos Aires, Hospital Thompson, San Martõ Ân, Prov. Buenos Aires, and Pharmacia Oncology, Buenos Aires, Argentina Objective To investigate Ki-67 and p21 Waf1/Cip1 expres- sion and apoptosis, before and after treatment, in tumour biopsies obtained from patients with super- ®cial bladder cancer who underwent vinorelbine intravesical therapy. Patients and methods Twenty patients with high-risk super®cial bladder cancer (including one or more of the following parameters: tumour diameter >3 cm, histological grade 3, or multicentric tumours) were treated 1±6 times (weekly) with intravesical vinor- elbine (50 mg/mL) instillations. Transurethral tumour marker biopsies were obtained one week before the ®rst instillation of the drug and one week after the last. The biopsies were immunostained for Ki-67 and p21 Waf1/Cip1 with monoclonal antibodies, on tissue sections derived from paraf®n-embedded samples obtained before and after vinorelbine treatments. In addition, apoptosis was determined using a terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labelling (TUNEL) technique. Results There were no signi®cant differences in the cell proliferation marker Ki-67 in biopsies taken before or after treatment. However, p21 Waf1/Cip1 showed signi®- cantly higher expression in biopsies obtained after vinorelbine treatment, with median (range) values of 40 (20±90)% before and 70 (50±80)% after (P<0.001, paired nonparametric Wilcoxon test). The apoptotic index was signi®cantly higher after vinorelbine therapy, with median (range) values of 0.89 (0.06±3.8)% before and 2.25 (0.17±18.7)% after treatment (P<0.001, paired nonparametric Wilcoxon test). Despite the brief treatment and few patients there was a clinical response in nine patients, together with low toxicity in all. Conclusion The intravesical treatment of tumours with vinorelbine affects p21 Waf1/Cip1 expression without blocking cell proliferation, although increasing apoptosis. The preliminary results suggest that vinorelbine may be useful for treating super®cial bladder tumours, and thus a phase II study is warranted. Keywords vinorelbine, bladder neoplasms, chemo- therapy Introduction Super®cial bladder cancer, comprising nearly 70% of all bladder carcinomas, is characterized by a high tumour recurrence after transurethral resection. Besides local surgical extirpation, a variety of intravesical chemo- therapies (e.g. doxorubicin, mitomycin or thiotepa) and immunotherapy (e.g. BCG) are used [1]. How- ever, most of these treatments are not curative and the identi®cation of more effective and less toxic therapies for recurrent bladder cancer would be highly advantageous. Vinorelbine, a semisynthetic Vinca alkaloid, has con®rmed activity in a variety of tumours, including non-small cell lung cancer and advanced breast cancer [2,3]. The drug induces not only mitotic arrest but also apoptosis, as shown in some tumour cell lines [4,5]. However, there are few or no data on the use of this drug in the clinical treatment of super®cial blad- der cancer. As the most common dose-limiting side- effects of vinorelbine, when administered systemically, are leukopenia and granulocytopenia, intravesical Accepted for publication 31 May 2001 BJU International (2001), 88, 425±431 # 2001 BJU International 425