Elevated apoptosis and cytokine markers in the urine of patients with carcinoma in situ of the bladder following treatment with a mycobacterial cell wall complex (MCC): correlation with clinical outcome.

NC Phillips, Ph.D.a, A Morales, M.D. b, S Reader, Ph.D. a, B. Filion, M.Sc. a, MC Filion, Ph.D. a.

a Bioniche Life Sciences Inc., Montreal, Quebec, Canada, b Kingston General Hospital, Kingston, Ontario, Canada.

AIM: Mycobacterial cell wall complex (MCC), a cell wall composition from Mycobacterium phlei, has cytokine stimulating and apoptosis-inducing activities. This study correlated MCC-induced cytokine stimulation and apoptosis with clinical response in patients with carcinoma in situ (CIS) of the bladder who had failed conventional treatment. METHODS: Cytokine synthesis was determined using PBMC’s, and cell cycle arrest and apoptosis induction (caspase-3 activation, DNA fragmentation, PARP and NuMA degradation) using 9 human bladder cancer cell lines. Seventeen patients with CIS who had failed BCG immunotherapy and/or chemotherapy were treated intravesically with MCC emulsion (4 mg) once weekly for 6 weeks, followed by 3 weekly treatments at 3 and 6 months. Urine samples were collected before and at 3 and 6 weeks treatment. Urinary IL-6, IL-8, IL-12, IL-18 (cytokines) and NuMA (apoptosis marker) were determined by ELISA, normalized to creatinine, and correlated with clinical outcome at 26 weeks. RESULTS: MCC stimulated IL-6, IL-8, IL-12 and IL-18 synthesis by PBMC’s, and caused cell cycle arrest (G0/G1) and apoptosis activation in the bladder cancer cell lines. IL-6 and IL-8 urine levels were elevated in the majority of patients treated with MCC emulsion (75% and 85% respectively). IL-12 and IL-18 urine levels were elevated in 73% and 57% of the patients respectively. NuMA was elevated in 47% and 33% of the patients. A complete clinical response at 26 weeks correlated with elevated IL-12, IL-18 and NuMA levels at 6 weeks (p<0.05), but not with elevated IL-6 or IL-8 levels. Patients failing MCC therapy showed no elevation of NuMA. CONCLUSIONS: MCC stimulates monocyte-associated cytokine synthesis and induces apoptosis in bladder cancer cell lines. This dual mode of action was confirmed clinically in patients with CIS refractory to treatment. Urine analysis of cytokines and apoptosis markers at 6 weeks treatment, which identified 80% of responding patients and all non-responding patients, may therefore be of use in predicting treatment outcome.

KEY WORDS: Bladder cancer, carcinoma in situ, apoptosis, cytokines, mycobacterial cell wall complex, therapy prediction.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Apoptosis - Molecular Mechanisms.