The effect of Keyhole Limpet Hemocyanin (KLH) on the rat bladder

Summary Immunostimulation with agents such as BCG or KLH may represent an adjuvant therapy in treating patients with bladder cancer. To investigate the effects of direct instillation and injection of KLH into the bladder of sensitized and nonsensitized rats, KLH was injected submucosally into the bladder. Histologically, a marked inflammatory reaction was seen without ulcerations.KLH seems to give a more predictable reaction without the BCG side effects. Intralesional application of KLH, which is now in a clinical trial, may play a role in the treatment of bladder cancer after transurethral resection of the tumor.     

The effect of intravesical Bacillus Calmette-Guerin instillations on the expression of inducible nitric oxide synthase in humans.

The activation of the inducible isoform of nitric oxide synthase (NOS) is associated with the production of large quantities of nitric oxide in response to cytokine stimulation. Bacillus Calmette-Guerin (BCG) mode of action against bladder carcinoma remains unclear, although a plethora of local and systemic events may follow its intravesical instillation. The present study was designed to investigate the expression of inducible NOS in normal and neoplastic urothelium and its alteration following tumor resection and subsequent intravesical immunotherapy. Bladder carcinoma and autologous normal bladder tissue specimens were procured from 36 patients undergoing transurethral resection. Tissue specimens were obtained from the same patients at first cystoscopy following six weekly intravesical instillations. Inducible NOS protein expression was assessed by immunohistochemistry in all tissue specimens. Immunostaining of normal urothelium for iNOS before treatment was negative in all but four cases. BCG treatment induced iNOS expression in tumor-free bladder tissue in 24 cases (66.6%). There were only four early tumor recurrences; interestingly, they corresponded to the cases with tumor cells expressing iNOS before BCG treatment, while novel tumors were also iNOS immunoreactive. BCG upregulated iNOS expression in normal human urothelial cells in vivo suggesting a role for nitric oxide in BCG mediated antitumor activity. Inducible NOS was detected in certain tumor specimens before and after BCG treatment implying a possible involvement in pro-tumor action.     

A mathematical model of combined bacillus Calmette-Guerin (BCG) and interleukin (IL)-2 immunotherapy of superficial bladder cancer

We report a mathematical model that describes the growth of superficial bladder cancer and the effect thereupon of immunotherapy based on the administration of Bacillus Calmette-Guerin (BCG) combined or not with interleukin-2 (IL-2). Intravesical instillations of BCG performed after surgical removal of tumors represents an established treatment with approximately 50% success rate. So far, attempts to improve this efficiency have not led to essential changes. However, convincing clinical results have been reported on the combination of IL-2 to BCG, even though this is still not applied in current practice. The present model provides insights into the dynamical outcomes arising in the bladder from the interactions of immune cells with tumor cells in the course of BCG therapy associated or not with IL-2. Specifically, from the simulations performed using seven ordinary and non-linear differential equations we obtained indications on the conditions that would result in successful bladder cancer treatment. We show that immune cells -effector lymphocytes and antigen-presenting cells-expand and reach a sustainable plateau under BCG treatment, which may account for its beneficial effect, resulting from inflammatory “side-effects” which eliminate residual or eventual newly arising tumor cells, providing thus protection from further cancer development. We find, however, that IL-2 does not actually potentiate the effect of BCG as regards tumor cell eradication. Hence, associating both under the conditions simulated should not result in more efficient treatment of bladder cancer patients.     

Mathematical Model of Pulsed Immunotherapy for Superficial Bladder Cancer

We present a theoretical study of superficial bladder cancer growth and its treatment via pulsed immunotherapy with Bacillus Calmette–Guérin (BCG), an attenuated strain of Mycobacterium bovis. BCG pulsed immunotherapy is a clinically established procedure for the treatment of superficial bladder cancer. In this paper, periodic BCG instillations are modeled using impulsive differential equations, which are studied using a combination of analytical and numerical techniques. In this way, we determine critical threshold values of the BCG instillation dose and rate of pulsing for successful treatment. We also identify treatment regimes in which tumor destruction occurs, but undesirable side effects are maintained at low levels by the immune system.     

Mathematical Model of BCG Immunotherapy in Superficial Bladder Cancer

Immunotherapy with Bacillus Calmette–Guérin (BCG)—an attenuated strain of Mycobacterium bovis (M. bovis) used for anti tuberculosis immunization—is a clinically established procedure for the treatment of superficial bladder cancer. However, the mode of action has not yet been fully elucidated, despite much extensive biological experience. The purpose of this paper is to develop a first mathematical model that describes tumor-immune interactions in the bladder as a result of BCG therapy. A mathematical analysis of the ODE model identifies multiple equilibrium points, their stability properties, and bifurcation points. Intriguing regimes of bistability are identified in which treatment has potential to result in a tumor-free equilibrium or a full-blown tumor depending only on initial conditions. Attention is given to estimating parameters and validating the model using published data taken from in vitro, mouse and human studies. The model makes clear that intensity of immunotherapy must be kept in limited bounds. While small treatment levels may fail to clear the tumor, a treatment that is too large can lead to an over-stimulated immune system having dangerous side effects for the patient.     

Urinary interleukin-2 may predict clinical outcome of intravesical bacillus Calmette-Guérin immunotherapy for carcinoma in situ of the bladder

PURPOSE: The mechanism by which bacillus Calmette-Guérin (BCG) mediates antitumor activity has not been clearly established. Specific cytokines in the urine after BCG intravesical instillation therapy may serve as a prognostic factor of treatment response. In this study, various urinary cytokines such as interleukin-1beta (IL-1beta), IL-2, IL-6, IL-8. IL-10, IL-12, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) were measured. MATERIALS AND METHODS: In total 20 patients were treated with BCG intravesical instillation therapy for carcinoma in situ of the bladder. At the completion of the first and eighth instillations, spontaneously voided urine specimens were collected before BCG instillation, every 2 h until 12 h, and thereafter until 24 h. All specimens were ultrafiltrated using an ADVANTEC UK-10 membrane. The cytokines were measured using ELISA and RIA techniques. RESULTS: Significantly higher levels of IL-2, IL-6, IL-8, IL-10, IFN-gamma, and TNF-alpha were detected in the eighth instillation as compared to the first instillation ( p<0.001). After BCG intravesical instillation therapy, treatment failure occurred in 6 of the 20 patients (30%), including primary failure (persistence of CIS) in 3, and de novo failure (tumor recurrence) in 3 with a median follow-up of 46.9 months. Significantly higher production of IL-2, IL-6, IL-8, IL-10, and TNF-alpha was observed in the responder group than in the non-responder group ( p<0.05). Multivariate analysis revealed IL-2 as an independent prognostic cytokine of responder status. CONCLUSIONS: This study indicates that urinary IL-2 at the eighth instillation of BCG may serve as a valuable prognostic factor of treatment efficacy as well as tumor recurrence after treatment.     

Superficial bladder cancer therapy

Bladder cancer treatment remains a challenge despite significant improvements in preventing disease progression and improving survival. Intravesical therapy has been used in the management of superficial transitional cell carcinoma (TCC) of the urinary bladder (i.e. Ta, T1, and carcinoma in situ) with specific objectives which include treating existing or residual tumor, preventing recurrence of tumor, preventing disease progression, and prolonging survival. The initial clinical stage and grade remain the main determinant factors in survival regardless of the treatment. Prostatic urethral mucosal involvement with bladder cancer can be effectively treated with Bacillus Calmette-Guerin (BCG) intravesical immunotherapy. Intravesical chemotherapy reduces short-term tumor recurrence by about 20%, and long-term recurrence by about 7%, but has not reduced progression or mortality. Presently, BCG immunotherapy remains the most effective treatment and prophylaxis for TCC (Ta, T1, CIS) and reduces tumor recurrence, disease progression, and mortality. Interferons, Keyhole-limpet hemocyanin (KLH), bropirimine and Photofrin-Photodynamic Therapy (PDT) are under investigation in the management of TCC and early results are encouraging. This review highlights and summarizes the recent advances in therapy for superficial TCC.     

Immune reactions in patients with superficial bladder cancer after intradermal and intravesical treatment with bacillus Calmette-Guérin

Summary The immune reactivity of patients with strongly recurrent superficial bladder cancer was followed after combined intravesical and intradermal bacillus Calmette-Guérin (BCG) immunotherapy. All patients in this study were previously treated without success with intravesical chemotherapy. The BCG treatment regimen consisted of weekly administrations with BCG (RIVM) for six consecutive weeks, both intravesically and intradermally. In this study, sera and peripheral blood leukocytes (PBL) of patients were tested serially. Besides BCG-antigen-specific reactions, e.g. skin reactivity to purified protein derivatives of Mycobacterium tuberculosis (PPD), antibody formation and antigen stimulation of PBL in vitro, non-antigen-specific immune reactivities were also measured, e.g. mitogen response and spontaneous cytotoxic activity of PBL. In addition the antibody response to bladder carcinoma antigens and the cytotoxic activity of PBL for the bladder carcinoma cell line T24 and the natural-killer-sensitive K562 cell line were investigated. The results obtained from the various assays were evaluated for their prognostic value in relation to the length of the tumor-free interval after the BCG treatment. Because sera and PBL were only obtained during the first 6 months after the BCG treatment, the immune reactivity was compared to the clinical results at that same time. At 6 months after therapy 12 out of 40 BCG-treated patients were tumor-free whereas 28 out of 40 showed a recurrence. Skin reactivity to tuberculin PPD was measured in 40 patients during a period of 3–6 months after therapy. Of patients who showed a recurrence of the tumor within 6 months, 48% of them showed a transient response or developed no response at all to PPD. In the group of patients with a longer tumor-free period (n=10), only one patient lost the response to tuberculin PPD. Although PBL of a limited number of patients were tested, it was observed that the cytotoxicity to the bladder carcinoma cell line T24, and the natural-killer-sensitive K562 cell line increased in a number of the patients (7 out of 14, and 9 out of 14 respectively). Reactivity of PBL to mitogens and subset distribution (ratio T-helper: T-suppressor/cytotoxic) were not influenced by the BCG treatment. Antibody response to mycobacterial antigen was detected in 9 out of 23 patients investigated. Of these 9 patients, 8 belonged to the group with a recurrence of the tumor within 6 months (n=17). There was no correlation between the skin reactivity and the antibody response to tuberculin PPD. Furthermore, none of the 25 patients showed an antibody response to bladder carcinoma antigens. Sera of bladder carcinoma patients (n=19) reduced the mitogen-induced proliferation of lymphocytes, compared to sera of healthy controls (n=13), indicating the presence of circulating suppressor factor(s). Our results indicate that the absence of a Mantoux conversion or the presence of transient reaction to tuberculin PPD were highly related (91%) to a relapse of the disease. On the other hand, the cytotoxic activity of PBL to T24 and K562 cell lines, or their reactivity to tuberculin PPD or mitogens, gives no predictive information about the clinical results (tumor-free interval) of the BCG therapy. Abbreviations used: BCG, bacillus Calmette-Guérin; NK, natural killer; PBL, peripheral blood leukocytes; PPD, purified protein derivative of Mycobacterium tuberculosis; ELISA, enzyme-linked immunosorbent assay     

Intracavitary Immunotherapy and Chemotherapy for Upper Urinary Tract Cancer: Current Evidence

A review of the literature was performed to summarize current evidence regarding the efficacy of topical immunotherapy and chemotherapy for upper urinary tract urothelial cell carcinoma (UUT-UCC) in terms of post-treatment recurrence rates. A Medline database literature search was performed in March 2012 using the terms upper urinary tract, urothelial cancer, bacillus Calmette-Guérin (BCG), and mitomycin C. A total of 22 full-text articles were assessed for eligibility, and 19 studies reporting the outcomes of patients who underwent immunotherapy or chemotherapy with curative or adjuvant intent for UUT-UCC were chosen for quantitative analysis. Overall, the role of immunotherapy and chemotherapy for UUT-UCC is not firmly established. The most established practice is the treatment of carcinoma in situ (CIS) with BCG, even if a significant advantage has not yet been proven. The use of BCG as adjuvant therapy after complete resection of papillary UUT-UCC has been studied less extensively, even if recurrence rates are not significantly different than after the treatment of CIS. Only a few reports describe the use of mitomycin C, making it difficult to obtain significant evidence.Key words: Upper urinary tract, Urothelial cell carcinoma, Bacillus Calmette-Guérin, Mitomycin C, Chemotherapy, ImmunotherapyAccording to the 2011 update of the European Guidelines for the diagnosis and management of upper urinary tract urothelial cell carcinoma (UUT-UCC),¹ urothelial carcinomas are the fourth most common tumors after prostate and breast cancer, lung cancer, and colorectal cancer. Bladder tumors account for 90% to 95% of urothelial carcinomas; UUT-UCC are relatively uncommon and account for only 5% to 10% of urothelial carcinomas. The annual incidence of UUT-UCC in Western countries is approximately one or two new cases per 100,000 inhabitants. Pyelocaliceal tumors are approximately twice as common as ureteral tumors. In 8% to 13% of cases, concurrent bladder cancer is present, and 60% of UUT-UCC are invasive at diagnosis, compared with only 15% of bladder tumors. This kind of carcinoma has a peak incidence in people in their 70s and 80s, with a higher prevalence in men.Radical nephroureterectomy (RNU) with excision of the bladder cuff represents the gold standard treatment for UUT-UCC, regardless of the location of the tumor in the upper urinary tract.¹ Lymph node dissection associated with RNU is of therapeutic interest and allows for optimal staging of the disease.Conservative surgery for low-risk UUT-UCC allows for preservation of the upper urinary renal unit; conservative management can be considered in imperative cases (renal insufficiency, solitary functional kidney) or in elective cases (ie, when the contralateral kidney is functional) for low-grade, low-stage tumors. Endoscopic ablation can be considered if a flexible ureteroscope, laser generator, and pliers (pluck) for biopsies are available, if the patient is informed of the need for closer follow-up, and if a complete resection is advocated.Segmental ureteral resection with wide margins provides adequate pathologic specimens for definitive staging and grade analysis while also preserving the ipsilateral kidney. Segmental resection is possible for the treatment of low- and high-risk tumors of the distal ureter, whereas segmental resection of the iliac and lumbar ureter is associated with a greater failure rate. Open resection of tumors of the renal pelvis or calices has almost disappeared.Percutaneous management can be considered for low-grade or noninvasive UUT-UCC that are inaccessible or difficult to manage by ureteroscopy, even if a theoretical risk of seeding exits in the puncture tract and if perforations occur during the procedure.After conservative treatment of UUT-UCC or for the treatment of carcinoma in situ (CIS), the instillation of bacillus Calmette-Guérin (BCG) or mitomycin C (MMC) is technically feasible by means of a percutaneous nephrostomy or even through a ureteric stent.Different agents have been used for topical therapy, including BCG, MMC, epirubicine, and thiotepa. Topical chemotherapeutic agents can be administered after endoscopic management, whereas instillations of BCG need to be postponed until the urothelium heals to avoid systemic side effects.According to a recent review,² topical therapy appears to be safe, although its efficacy is debatable. Complications from the administration of topical immunotherapy or chemotherapy can be avoided by maintaining low intracavitary pressures during administration. Renal function does not seem to be impaired after instillation of BCG or MMC.³ No systemic side effects result from perfusion with MMC, and persistent fever was reported in 5% of patients in combined major series after BCG administration; therefore, this side effect was resolved with appropriate antimicrobial therapy in all cases. Furthermore, up to 25% of patients may have granulomatous involvement of the urinary tract after BCG.This review summarizes current evidence about the efficacy of topical immunotherapy and chemotherapy in terms of post-treatment recurrence rates.     

Cytokine gene polymorphisms can alter the effect of Bacillus Calmette-Guérin (BCG) immunotherapy

Various types of cancer are more frequent in men than women, and bladder cancer is one of the most common of these. Intravesical instillation of Bacillus Calmette-Guérin (BCG) after transurethral resection is the most effective treatment for superficial bladder cancers. The main aim of this study was to investigate for possible links between cytokine gene polymorphisms and different outcomes after BCG immunotherapy. Sixty patients who had been diagnosed with transitional cell cancer were investigated. All genotyping experiments were performed using polymerase chain reaction sequence-specific primers and a commercially available kit. The genes investigated were those that code for interleukin (IL)-1alpha, IL-1beta, IL-1R, IL-1RA, IL-4RA, IL-2, IL-4, IL-6, IL-10, IL-12, interferon-gamma (IFN-gamma), transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha). Analyses of the data identified TGF-beta codon 25 GG (92.85% vs. 64.44%, p=0.04, OR=7.17), IL-4 -1098 GG (16.6% vs. 0.0%, p=0.05, OR=18.33), IL-10 -1082 GG (28.5% vs. 6.8%, p=0.05, OR=5.47), and IL-10 -1082 GCC/GCC (28.57% vs. 4.5%, p=0.025, OR=8.4) polymorphisms as risk factors for progression of bladder cancer.     

Auxotrophic recombinant Mycobacterium bovis BCG overexpressing Ag85B enhances cytotoxicity on superficial bladder cancer cells in vitro

BCG therapy remains at the forefront of immunotherapy for treating patients with superficial bladder cancer. The high incidence of local side effects and the presence of non-responder diseases have led to efforts to improve the therapy. Hence, we proposed that an auxotrophic recombinant BCG strain overexpressing Ag85B (BCG ?leuD/Ag85B), could enhance the cytotoxicity to the human bladder carcinoma cell line 5637. The rBCG was generated using an expression plasmid encoding the mycobacterial antigen Ag85B to transform a BCG ?leuD strain. The inhibitory effect of BCG ?leuD/Ag85B on 5637 cells was determined by the MTT method, morphology observation and a LIVE/DEAD assay. Gene expression profiles for apoptotic, cell cycle-related and oxidative stress-related genes were investigated by qRT-PCR. Bax, bcl-2 and p53 induction by BCG ?leuD/Ag85B treatment was evaluated by Western blotting. BCG ?leuD/Ag85B revealed a superior cytotoxicity effect compared to the control strains used in this study. The results showed that the expression level of pro-apoptotic and cell cycle-related genes increased after BCG ?leuD/Ag85B treatment, whereas the mRNA levels of anti-apoptotic genes decreased. Interestingly, BCG ?leuD/Ag85B also increased the mRNA level of antioxidant enzymes in the bladder cancer cell line. Bax and p53 proteins levels increased following treatment. In conclusion, these results suggest that treatment with BCG ?leuD/Ag85B enhances cytotoxicity for superficial bladder cancer cells in vitro. Therefore, rBCG therapy may have potential benefits in the treatment of bladder cancer.     

Bacillus Calmette Guerin induces fibroblast activation both directly and through macrophages in a mouse bladder cancer model

Background

Bacillus Calmette-Guerin (BCG) is the most effective treatment for non-muscle invasive bladder cancer. However, a failure in the initial response or relapse within the first five years of treatment has been observed in 20% of patients. We have previously observed that in vivo administration of an inhibitor of nitric oxide improved the response to BCG of bladder tumor bearing mice. It was described that this effect was due to a replacement of tumor tissue by collagen depots. The aim of the present work was to clarify the mechanism involved in this process.

Methodology/Principal Findings

We demonstrated that BCG induces NIH-3T3 fibroblast proliferation by activating the MAPK and PI3K signaling pathways and also differentiation determined by alpha-smooth muscle actin (alpha-SMA) expression. In vivo, intratumoral inoculation of BCG also increased alpha-SMA and collagen expression. Oral administration of L-NAME enhanced the pro-fibrotic effect of BCG. Peritoneal macrophages obtained from MB49 tumor-bearing mice treated in vivo with combined treatment of BCG with L-NAME also enhanced fibroblast proliferation. We observed that FGF-2 is one of the factors released by BCG-activated macrophages that is able to induce fibroblast proliferation. The involvement of FGF-2 was evidenced using an anti-FGF2 antibody. At the same time, this macrophage population improved wound healing rate in normal mice and FGF-2 expression was also increased in these wounds.

Conclusions/Significance

Our findings suggest that fibroblasts are targeted by BCG both directly and through activated macrophages in an immunotherapy context of a bladder murine model. We also described, for the first time, that FGF-2 is involved in a dialog between fibroblasts and macrophages induced after BCG treatment. The fact that L-NAME administration improves the BCG effect on fibroblasts, NO inhibition, might represent a new approach to add to the conventional BCG therapy.     

Virus therapy for bladder cancer

Approximately 50,000 cases of superficial bladder cancer are diagnosed annually in the United States. Immunotherapy utilizing intravesical BCG is the most effective standard therapy for superficial transitional cell carcinoma of the bladder. Based on ease of administration, limited systemic dissemination, and the demonstrated activity of immunotherapy, superficial bladder cancer is an excellent target for virus based gene and immunotherapy. Thus far, clinical trials of virus therapy for bladder cancer have yielded mixed results. In this paper the results of several virus based clinical trials for bladder cancer are reviewed.     

Presence of interleukin-2 in urine of superficial bladder cancer patients after intravesical treatment with bacillus Calmette-Guérin

Summary Urine samples were obtained from patients with superficial bladder cancer after immunotherapy with bacillus Calmette-Guérin (BCG). The patients were repeatedly (once a week for 6 consecutive weeks) treated with intravesical administration of approximately 5 × 10⁸ culturable particles of BCG. Some patients received more than six BCG instillations. The urine samples were investigated for the presence of interleukin-2 (IL-2) in an in vitro bioassay using a murine cytotoxic T cell line (CTTL-16) that shows IL-2-dependent growth. Preliminary experiments indicated the presence of inhibitory factors in the urine. This inhibitory activity was abolished after 24 h dialysis. In a neutralization assay with both polyvalent and monoclonal anti-(human IL-2) antibody it was demonstrated that there was indeed IL-2 in the urine samples. In 8 of 11 patients the presence of IL-2 in the urine was demonstrated. The IL-2 production was directly related to the BCG administration as samples obtained just before the BCG instillation were always negative. In IL-2-positive samples a maximum level of IL-2 was observed between 2 h and 6 h after the BCG instillation. In urine samples obtained 24 h after the BCG IL-2 was not detected. In most patients the urine became positive after the third or fourth BCG instillation     

Present status of BCG immunotherapy of malignant melanoma

Summary BCG systemic adjuvant immunotherapy may be effective for improving both the recurrence and survival rates in patients with regional metastases from malignant melanoma. Clinical trials show that many of the principles derived from the study of animal tumor systems are applicable to human cancer in that immunotherapy is most effective for a small residual number of tumor cells. BCG treatment fulfills many of the ideal criteria for adjuvant treatment following surgery when disease burden is lowest. It is relatively nontoxic; it is effective for disseminated melanoma; it has systemic activity in the adjuvant treatment of subclinical metastases. However, until clinical trials are complete, BCG adjuvant therapy must be considered investigational.Supported by USPHS grants CA05252, CA12582, and NIH 0732001 CB43852.     

A possible mechanism of intravesical BCG therapy for human bladder carcinoma: involvement of innate effector cells for the inhibition of tumor growth

Intravesical bacillus Calmette-Guerin (BCG) therapy is considered the most successful immunotherapy against solid tumors of human bladder carcinoma. To determine the actual effector cells activated by intravesical BCG therapy to inhibit the growth of bladder carcinoma, T24 human bladder tumor cells, expressing very low levels of class I MHC, were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) with live BCG. The proliferation of T24 cells was markedly inhibited when BCG-infected dendritic cells (DCs) were added to the culture although the addition of either BCG or uninfected DCs alone did not result in any inhibition. The inhibitory effect was much stronger when the DCs were infected with live BCG rather than with heat-inactivated BCG. The live BCG-infected DCs secreted TNF-α and IL-12 within a day and this secretion continued for at least a week, while the heat-inactivated BCG-infected DCs secreted no IL-12 and little TNF-α. Such secretion of cytokines may activate innate alert cells, and indeed NKT cells expressing IL-12 receptors apparently proliferated and were activated to produce cytocidal perforin among the PBMCs when live BCG-infected DCs were externally added. Moreover, depletion of γδ T-cells from PBMCs significantly reduced the cytotoxic effect on T24 cells, while depletion of CD8β cells did not affect T24 cell growth. Furthermore, the innate effectors seem to recognize MICA/MICB molecules on T24 via NKG2D receptors. These findings suggest the involvement of innate alert cells activated by the live BCG-infected DCs to inhibit the growth of bladder carcinoma and provide a possible mechanism of intravesical BCG therapy.     

Results of randomized trials of immunotherapy for acute leukemia

Summary For more than a decade clinical trials have attempted to define the role of immunotherapy in the treatment of patients with acute leukemia. Based on animal studies which indicated that non-specific immune stimulation had an antitumor effect if the tumor burden was small, the use of immunotherapy during remission in patients with acute leukemia seemed appropriate following the initial report of the success of bacillus Calmette-Guerin (BCG) in prolonging remission duration and survival in acute lymphoblastic leukemia. Therefore a series of randomized clinical trials was initiated to confirm these original observations. In four studies comparing BCG inoculations, with or without allogeneic leukemia cells, and chemotherapy or no therapy, no advantage of immunotherapy was noted. Immunotherapy appeared to be equally as good as chemotherapy. A combination of BCG and chemotherapy showed some advantage in one study, but no advantage was noted in two other studies.In acute myeloblastic leukemia several randomized trials suggested that BCG or one of its derivatives when given alone, in combination with allogeneic cells, or with chemotherapy had a marginal effect in prolonging remission duration and survival when compared to chemotherapy or rno therapy.In conclusion, immunotherapy during remission has marginal activity in acute leukemia.     

Induction of urinary interleukin-1 (IL-1), IL-2, IL-6, and tumour necrosis factor during intravesical immunotherapy with bacillus Calmette-Guérin in superficial bladder cancer

Summary To study the local immunological effects of intravesical bacillus Calmette-Guérin (BCG) therapy in superficial bladder cancer patients, the production of interleukin-1 (IL-1), IL-2, IL-6, tumour necrosis factor (TNF), and interferon (IFN) was investigated in the urine. Urine specimens were collected during the six weekly BCG instillations, before instillation, and 2, 4, 6, 8, and 24 h thereafter. Results were standardized to urine creatinine. In general, the concentration of IL-1 increased markedly during the first three BCG instillations, reaching a plateau from instillations 3 to 6. IL-2 was not detected after the first BCG instillation, but from the second instillation onwards the mean IL-2 concentration increased rapidly. With respect to IL-6, patients had relatively high levels in the urine after the first BCG instillation. A relatively moderate increase of the IL-6 concentration was observed during the following weeks. Like IL-2, TNF was only detected after repeated BCG instillations. Generally the highest TNF levels were found after BCG instillation 5. The presence of IFN could not be demonstrated. With respect to the occurrence of the cytokines during the first 24 h after the BCG instillation, TNF, IL-2, and IL-6 were detectable 2 h after the instillation. In contrast, IL-1 seemed to appear later, i.e. from 4 h onwards. TNF decreased most rapidly; it was nearly absent in 6-h samples. Generally IL-2 was not detectable in the 8-h samples, whereas IL-1 and IL-6 were present up to 8 h after instillation of BCG. The presence of TNF was found less frequently than the presence of IL-1, IL-2, and IL-6. Neutralization experiments indicated that most of the IL-1 present in the urine after BCG treatment was IL-1. In conclusion, activation of BCG-specific T cells was indicated by the detection of IL-2. The presence of IL-1, IL-6, and TNF might suggest activation of macrophages by intravesically administered BCG, although production by other cell types cannot be excluded. It is suggested that these cytokines, in combination with the leucocytes that are known to be recruited to the bladder in reaction to the BCG treatment, may play an important role in the antitumour activity of BCG against bladder cancer. For monitoring purposes, collection of urine might be performed during the first 6 h after BCG instillations 4–6. A correlation between the presence of cytokines in the urine and the clinical response has yet to be evaluated.     

Role of IL-12 in the induction and potentiation of IFN-gamma in response to bacillus Calmette-Guérin.

Although Mycobacterium bovis bacillus Calmette-Guérin (BCG) has been accepted as the most effective agent in clinical use against superficial bladder cancer, its mechanism of action remains incompletely understood. A kinetic analysis in assessing the potential role of cytokines from BCG-stimulated murine splenocytes showed that IL-12 expression preceded that of other cytokines. Experiments subtracting endogenous BCG-driven IL-12 using neutralizing Ab or augmenting its activity with supplemental rIL-12 revealed not only that IL-12 plays a dominant role in IFN-gamma induction but also that it is normally dose limiting. A striking increase in IFN-gamma production could be generated in both mouse and human immunocompetent cell culture by the addition of even a small amount of rIL-12. Moreover, this same synergistic effect could be replicated during in vivo administration of BCG plus rIL-12 into the mouse bladder and was observed in a patient receiving intravesical combination therapy. In costimulation cultures, this synergy appeared to partially rely on IL-18 and IL-2 and could be down-regulated by IL-10. This suggests that a dynamic interplay between Th1 and Th2 cytokines is responsible for net IFN-gamma production. The ability of supplemental exogenous IL-12 to strongly shift this balance toward Th1 provides an immunological basis for using it in conjunction with intravesical BCG for bladder cancer immunotherapy.     

Production of urinary tumour necrosis factors and soluble tumour necrosis factor receptors in bladder cancer patients afterbacillus Calmette-Guerin immunotherapy

Intravesical immunotherapy for bladder cancer is the most effective form of tumour immunotherapy. Following repeated instillations of bacillus Calmette-Guérin (BCG) organisms into the bladder large 0quantities of several cytokines are detected in the urine. These cytokines include interleukins IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumour necrosis factor α (TNFα), interferon γ (IFNγ) and also soluble intercellular adhesion molecule ICAM-1. In the work reported here we simultaneously quantified urinary levels of TNFα, TNFβ, TNF receptor I and TNF receptor II by enzyme-linked immunosorbent assay (ELISA) techniques and compared this with bioactive levels of TNF. This was undertaken with a limited number of patients throughout a course of six instillations of immuno therapy. Sequential instillations of BCG induced secretion of TNFα and TNFβ into urine. These cytokines were not always secreted simultaneously, perhaps suggesting differential regulation of their synthesis. Maximal concentrations of TNFα were 675 pg/ml and TNFβ 47 pg/ml. High levels of both species of soluble TNF receptor were readily identified in urine. Maximal levels of sTNF-RI were 6200 pg/ml (range from 0) and for sTNF-RII 7800 pg/ml (range from 0). Contrasting with earlier published observations concerning cytokine levels, the concentration of soluble receptor did not increase with repeated instillation. In apparent contrast with the ELISA data, very low levels of bioactive TNF were identified by the L929 bioassay (maximum concentration 1 U/ml) despite the elevated concen t ration of immunoreactive TNF. The large concentrations of soluble TNF receptor in patients’ urine samples could account for the apparently low bioactivity as determined by the L929 cytotoxicity assay. The precise nature of the role of TNF in BCG immunotherapy remains undetermined; however, it is thought that proinflammatory cytokines are in part responsible for the clinical efficacy of this therapeutic approach. Whether other cytokines are antogonised by soluble binding proteins remains to be determined. Furthermore, whether TNF is bioactive in the bladder wall and only neutralised in the urine also requires investigation. Received: 24 August 1994 / Accepted: 17 October 1994