Elizabeth C. de Boer

Urinary Cytokines During Intravesical Bacillus Calmetteguerin Therapy for Superficial Bladder Cancer: Processing, Stability and Prognostic Value

PURPOSE An accurate prognostic indicator to identify nonresponding patients with superficial transitional cell carcinoma of the bladder at an early stage of intravesical bacillus Calmette-Guerin (BCG) therapy is urgently needed. MATERIALS AND METHODS The processing conditions and stability of several BCG induced urinary cytokines were analyzed, as was the possible correlation between these cytokines (indicating immune responsiveness to BCG) and bladder tumor recurrence. We studied 23 patients with superficial transitional cell carcinoma of the bladder. Monitoring was performed by serial collection of urine during the first 24 hours after each of the 6 consecutive weekly intravesical BCG instillations. Baseline pre-therapy cytokine levels were 3.9 +/- 4.7 pg./mumol creatinine for interleukin-6 and 0.1 +/- 0.2 pg./mumol creatinine for tumor necrosis factor-alpha (all measured by enzyme-linked immunosorbent assay). To investigate the correlation between interleukin-2 and bladder tumor recurrence, patients were stratified into 2 groups based on an early (6 months or less) or late (greater than 6 months) recurrent tumor. For each patient the highest cytokine value measured during the 6-week BCG treatment course was evaluated. RESULTS The results were positive if the level in urine exceeded 0.34 units interleukin-2 per mumol. creatinine. A significant correlation between urinary interleukin-2 and tumor recurrence was found (p = 0.003, 23 patients). Of the studied cytokines obtained from BCG treated patients, interleukin-1 beta, 2 and 6 but not tumor necrosis factor-alpha were stable in urine at 4C and 20C. At 37C all cytokines were unstable. Interferon-gamma could only be detected in immediately dialyzed urine and its occurrence correlated most with that of interleukin-2. Processing of urine by centrifugation to remove leukocytes immediately after collection was not required for reliable measurements of interleukins-2 and 6. Based on these results interleukins-2 and 6 were preferred for extensive monitoring of the BCG induced immune reaction. CONCLUSIONS Our study provides significant evidence for a correlation between urinary cytokine induction and clinical response following intravesical BCG therapy. Particularly, monitoring of interleukin-2 may have the potential for prognostic value provided that strict precautions regarding urine collection, such as maximal 2-hour sampling and immediate cooling, are taken.

URINARY INTERLEUKIN-2 MONITORING DURING PROLONGED BACILLUS CALMETTE-GUERIN TREATMENT: CAN IT PREDICT THE OPTIMAL NUMBER OF INSTILLATIONS?

PURPOSE In patients with superficial bladder cancer treated with a first 6-week instillation course of bacillus Calmette-Guerin (BCG) the induction pattern of urinary interleukin (IL)-2 has been described, and the levels of urinary IL-2 were associated with the clinical response to BCG treatment. We evaluated urinary IL-2 kinetics in patients with recurrent superficial bladder tumor receiving a second or third 6-week BCG instillation course. To our knowledge there have been no studies of prolonged BCG treatment and urinary cytokine responses. MATERIALS AND METHODS Urinary IL-2 was determined in 12 patients with superficial transitional cell carcinoma of the bladder receiving a complete (6-week) second or third BCG instillation course and in 3 patients receiving 3 BCG instillations during a maintenance schedule at month 3. Urinary IL-2 was determined with an enzyme-linked immunosorbent assay using an oligoclonal system. RESULTS Of 12 patients 10 had a urinary IL-2 positive response during the subsequent BCG course and at week 1 urinary IL-2 was already increased. Comparing the urinary IL-2 kinetics observed during a second or third with a first course, urinary IL-2 tended to be higher during the first and lower during the last weeks. If the interval between subsequent courses was short (12 months or less) significantly higher urinary IL-2 levels at weeks 1 and 2, and a lower level at week 6 were observed. CONCLUSIONS During a repeat BCG instillation course urinary IL-2 reached a maximum at an earlier week, especially if the interval between the subsequent courses was short. Since an association between urinary IL-2 levels and response to BCG treatment during an induction course has been observed, these immunological data argue in favor of a limited number of instillations during prolonged BCG therapy which could reduce side effects as well as costs.

Urinary Cytokines Reflecting the Immunological Response in the Urinary Bladder to Biological Response Modifiers: Their Practical Use

Background: Intravesical bacillus Calmette-Guérin (BCG) immunotherapy is currently the most effective treatment for superficial transitional cell carcinoma (TCC) of the urinary bladder. In recent years, the substantial number of patients not responding to BCG or experiencing considerable toxicities has stimulated studies addressing either the development of improved BCG treatment schedules or the exploration of the therapeutic value of a series of (novel) biological response modifiers, like interferons (IFNs), interleukin (IL) 2 and keyhole limpet hemocyanin. Although the actual mechanism by which BCG exerts its antitumor effect still needs detailed unraveling, current available knowledge suggests the induction of a T helper 1 (Th1) or Th1-like cytokine profile, represented by IL-2, IL-12 and IFN-γ, as essential in the development of a cell-mediated antitumor activity. Conclusions: In this review, it is argued that incorporation of urinary cytokine determinations, like IL-2 and possibly IL-12 and IFN-γ, may represent a valuable approach in the optimization and individualization of the BCG therapy and an early, initial evaluation of the potential efficacy of novel immunomodulating agents in the treatment of superficial TCC.

Double fluorescent flow cytometric assessment of bacterial internalization and binding by epithelial cells

This study describes a new flow cytometric method for assessment of phagocytosis of specific bacteria (bacillus Calmette-Guérin (BCG) and Escherichia coli) by bladder epithelial cells. The internalization assay consisted of labeling bacteria chemically with fluorescein isothiocyanate (FITC). Subsequent to incubation of fluoresceinated bacteria with internalizing cells, adherent nonphagocytosed bacteria were marked by two-step labeling using specific antibodies and phycoerythrin (PE)-conjugated antibodies. Double fluorescent FACS analysis differentiated between bacterial phagocytosis and adherence. The validity of the method was shown by inhibition of BCG phagocytosis at 4 degrees C by cytochalasin B, by removal of excess free bacteria, and by anti-BCG antibodies. BCG-phagocytizing and -nonphagocytizing cell lines were discriminated by applying this technique to a series of bladder carcinoma cell lines. There seemed to be a relationship between phagocytic capacity and grade of differentiation in these cell lines, which may have implications for topical BCG immunotherapy in superficial bladder cancer. In conclusion, a new, reliable, rapid, and relatively simple double fluorescent method is described for quantification of specific bacterial internalization by large numbers of (bladder) epithelial cells. This method should be generally applicable to the study of in vitro interaction between bacteria and different types of host cells.

Immunostimulation in the urinary bladder by local application of Nocardia rubra cell-wall skeletons (Rubratin) and bacillus Calmette-Guérin as therapy for superficial bladder cancer : A comparative study

Twelve patients with superficial bladder cancer were treated with intravesical instillations of Rubratin (ASTA Pharma AG, Frankfurt, Germany), a cell-wall preparation of Nocardia rubra. The objective was to compare the immunostimulating effect of Rubratin with that of bacillus Calmette-Guérin (BCG). Local immunostimulation was determined by cytokine induction in serially collected urine samples during the first 24 h after each instillation, leukocyte influx into the urine, and phenotypic analysis of the lymphocyte fraction. Levels of Rubratin-induced interleukin (IL)-1 beta, IL-6, and tumor necrosis factor-alpha were significantly elevated compared with pretherapy levels. Rubratin induced leukocyte influx into the urine. T-cell activation (IL-2 receptor and human leukocyte antigen-DR expression) can be induced, and CD4:CD8 cell ratios can be increased. All parameters indicated that Rubratin-induced immunostimulation was less than that associated with BCG. In conclusion, although local Rubratin-induced immunostimulation occurs in a limited number of patients, the amount of immunocompetent cells attracted to the bladder seems to be less than that associated with BCG therapy, thus resulting in lower levels of cytokine production (which may reflect less clinical efficacy).

Retrovirus type C in the mouse bladder carcinoma cell line MBT-2.

PURPOSE The presence of replicating type C retrovirus in MBT-2 mouse bladder carcinoma cells is reported. This MBT-2 tumor cell line is nowadays globally distributed. The cells have been and are still used to study various aspects of bladder cancer. While studying the phagocytic capacity of MBT-2 cells for BCG organisms by electron microscopic methods, the presence of this retrovirus was noticed. MATERIALS AND METHODS MBT-2 cells that were cultured in vitro as well as cells from intravesically and intradermally grown MBT-2 tumors from syngeneic mice were investigated using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques. RESULTS All samples including the earliest generation MBT-2 cells that could be traced from stocks of other research groups contained the C type retrovirus, suggesting a contamination in all available generations of the MBT-2 cell line. CONCLUSIONS As this tumor cell line is widely used in immunologic studies of the response to bladder cancer, it is important to consider the possible presence of type C viruses and associated antigens, since they could contribute to or interfere with the responses being measured. Studies should be initiated to determine whether viral antigen expression is involved in the immune rejection of MBT-2 bladder cancer. As a consequence, clinical implementation of immunological treatment strategies should not be based on results obtained with the MBT-2 model alone, but preferably should be confirmed with other (bladder) carcinoma models.