Michael A. O'Donnell

BCG immunotherapy of bladder cancer: 20 years on

1The situation is similar for most industrialised western nations, with incidence rates of 18 to 30 new cases per 10 0 000 men placing bladder cancer among the top five cancers in this sex. It is unclear why women are affected a third to a quarter less often than men. In 75% of patients the disease is diagnosed in its early superficial stage, usually as a result of gross or microscopic blood in the urine. However, with an overall 65% recurrence rate and 30% progression rate, even these patients need lifelong medical vigilance involving periodic internal inspections of their bladders. The cause of bladder cancer is unknown, but the disease has been strongly associated with exposure to certain aromatic chemicals, notably aniline dyes and benzidine compounds. Cigarette smoking with its release of -naphthalene and -napthalene into the urine is estimated to cause over one half of bladder cancers found in men and one third of those found in women. 2 Over 90% of bladder cancers found in such settings are of the transitional cell type, with squamous cell carcinoma usually occuring in patients with chronic infections such as bilhariasis. Among the transitional cell carcinomas, depth of penetration (stage) and degree of cellular anaplasia (grade) are the most important factors for prognosis. One particular mucosal high-grade lesion called carcinoma-in-situ is not surgically accessible because of its diffuse surface-spreading behaviour. Untreated carcinoma-in-situ will progress to muscleinvasive disease in about 80% of cases within 5 years. 3

SUPERFICIAL BLADDER CANCER: THE ROLE OF INTERFERON-alpha

PURPOSEnWe evaluate the clinical experience with recombinant interferon-alpha in superficial transitional cell carcinoma and discuss the most rational use of recombinant interferon-alpha in the context of current treatment options.nnnMATERIALS AND METHODSnThe available data were reviewed and discussed at a consensus conference in August 1996. The conclusions and recommendations are those of the authors based on the consensus reached at that meeting.nnnRESULTSnWhile bacillus Calmette-Guerin (BCG) is recognized as the most efficacious intravesical agent in the prophylaxis and treatment of superficial transitional cell carcinoma, it is associated with significant toxicities and a 20 to 40% relapse rate. Interferons, particularly recombinant interferon-alpha, have demonstrated efficacy against primary and recurrent papillary transitional cell carcinoma and carcinoma in situ with minimal toxicity, although the response and relapse rates are inferior to BCG. Intravesical recombinant interferon-alpha therapy has also produced responses in patients who failed to respond or were refractory to BCG or chemotherapy.nnnCONCLUSIONSnThe clinical experience suggests that recombinant interferon-alpha has an important role in the treatment of superficial transitional cell carcinoma, particularly as second line therapy following failure of BCG or chemotherapy, and it may have synergistic effects when combined with chemotherapy or BCG. We propose a prospective randomized study comparing the efficacy of recombinant interferon-alpha, BCG and BCG plus recombinant interferon-alpha as maintenance following complete response to primary BCG therapy. The proposed study would also investigate the efficacy of BCG plus recombinant interferon-alpha as second line therapy following BCG failure. This study will be important to determine the most effective strategy to integrate recombinant interferon-alpha into current treatment options for superficial bladder cancer.

Recombinant bacille Calmette-Guérin (BCG) expressing human interferon-alpha 2B demonstrates enhanced immunogenicity.

To increase its immunostimulatory properties, BCG was genetically engineered to secrete recombinant human interferon‐alpha 2B (rhIFN‐α) under control of the mycobacterial heat shock protein (hsp)60 promoter and the α antigen signal sequence. Expression of rhIFN‐α was readily detectable by ELISA and on Western blotting. When compared with control BCG, rhIFN‐α BCG was substantially more active in inducing the production of IFN‐γ and IFN‐inducible protein 10 (IP‐10) from human peripheral blood mononuclear cells, while IL‐10 production was correspondingly decreased. These effects were reversible upon antibody neutralization of rhIFN‐α. Among 10 patients tested, rhIFN‐α BCG enhanced IFN‐γ production in all patients ranging from 1·4‐ to 23·7‐fold with a general trend toward greatest enhancement among those with weakest baseline responses to control BCG. Correspondingly, rhIFN‐α BCG decreased IL‐10 production in all patients by 1·2–4·8‐fold. The onset of IFN‐γ production induced by rhIFN‐α BCG was also more rapid, occurring within 4u2003h after stimulation versus >u200a24u2003h with wild‐type BCG. The observation that the maximum IFN‐γ induction depends on the simultaneous presence of both IFN‐α and BCG highlights the advantages of rhIFN‐α BCG. Taken together, these immunostimulatory properties of rhIFN‐α BCG suggest that it may be a superior agent for immunotherapeutic protocols involving live BCG in humans.

Bacillus Calmette-Guérin immunotherapy for superficial bladder cancer. New prospects for an old warhorse.

The use of live M. bovis BCG to treat superficial bladder cancer has endured its seemingly anachronistic origin in the early days of tumor immunology to emerge as the therapy of choice for superficial bladder cancer. Its superiority over conventional intravesical therapy has been established for tumor prophylaxis and treatment of residual disease and CIS, providing long-term results that have translated into improvements in disease progression and survival. Although its exact mechanism of action remains elusive, extensive studies suggest that this intracellular pathogen stimulates the immune system to produce powerful cytokine mediators and effector cells that act locally to destroy bladder tumors. Although unique toxicities occur by virtue of BCGs use as a live vaccine, this same liability has opened the door for new opportunities through the use of recombinant DNA technology. Exciting prospects include the use of BCG as a cytokine carrier and as a tumor antigen depot. Genetic engineering may also yield varients that are both intrinsically safer and more specific in bladder tumor targeting.

Co-expression of interleukin-2 and green fluorescent protein reporter in mycobacteria: in vivo application for monitoring antimycobacterial immunity

Recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing green fluorescent protein (rBCG-GFP), driven by the mycobacterial heat shock protein 70 (HSP 70) promoter from an autonomously replicating plasmid, was genetically engineered to co-express mouse interleukin-2 (IL-2) by introduction of an independent HSP 60 promoter. To monitor host antimycobacterial immunity, C57BL/6 mice were intravenously infected with IL-2 expressing and non-expressing GFP rBCGs. Both rBCGs were clearly imaged and easily quantified with ultraviolet microscopy of tissue sections and whole organ suspensions. Enhanced mycobacterial clearance from the spleens of mice infected with the rBCG-IL-2/GFP strain was apparent by both diminished bacterial counts and spleen weights during the first 6 weeks post-infection relative to rBCG-GFP. T helper type 1 (TH1) cytokine production and proliferative response to BCG restimulation was also elevated from in vitro splenocyte cultures taken from the rBCG-IL-2/GFP-infected group. Taken together, these results suggest that IL-2 expression from rBCG augmented host protective immunity to mycobacterial infection via an enhanced TH1 immune response. Mycobacterial expression vectors that allow simultaneous but independent production of reporter proteins and bioactive substances provide an ideal means for monitoring the in vivo fate of recombinant mycobacteria.

Evidence-based illiteracy: time to rescue "the literature"

That oft-evoked entity “the literature” has, I submit, been corrupted by a language created by people who write not to be read but to be cited. We live in a world that equates data with knowledge. Medicine may have moved from mystic certainty towards scientific uncertainty, yet many of its practitioners still behave as if Galileo had never lived. Wedded to the inductive approach of Francis Bacon, they choose to act like vacuum cleaners, hoovering up vast quantities of data. At the same time, we have created a monstrous regiment of journals to cater for people eager to see their names in print not because they are bursting to tell us something but for more solemn reasons. Another paper means another line on a curriculum vitae, another step towards a job or a research grant. In 1976, we missed one of the great opportunities of 20th-century medicine when a Dublin physician J B Healy wrote to this journal and, like another Irishman 250 years before him, submitted a modest proposal:

The genetic reconstruction of BCG as a new immunotherapeutic tool

The bacillus of Calmette and Guérin (BCG), long appreciated for its role as a live vaccine for the prevention of tuberculosis, is undergoing a rebirth as a recombinant delivery vehicle for foreign antigens and bioactive proteins. Recombinant BCG causes long-lived specific humoral and cellular immunity and may ultimately prove to be a powerful and cost-effective new weapon against both infectious pathogens and certain cancers.

INTERLEUKIN-12: Opportunities for the Treatment of Bladder Cancer

Despite incomplete understanding of the human immune system, the rapid progress in tumor immunology provides a framework for more effective and safe interventions in the near future. Early approaches in patients with cancer that have focused on the nonspecific and broad stimulation of the immune system by interferons and IL-12 will be replaced by the highly specific stimulation of immune reactions targeting precisely defined tumor antigens. IL-12 has several biologic properties that seem useful in immune therapy for bladder cancer. The striking antitumor responses with IL-12 in preclinical animal models of bladder cancer provide optimism that future clinical trials involving this agent may impact on the risk and mortality associated with this disease.

PHARMACOLOGICAL BIOCOMPATIBILITY BETWEEN INTRAVESICAL PREPARATIONS OF BCG AND INTERFERON-ALPHA 2B

PURPOSEnTo determine if BCG and interferon alpha-2B are mutually compatible as mixed intravesical agents for clinical bladder cancer therapy.nnnMATERIALS AND METHODSnMutual compatibility was assessed by measuring IFN-alphas effect on BCG metabolic activity, growth rate, and clumping tendency and conversely by observing BCGs effect on IFN-alphas anti-viral activity. Optical density at 600 nm. (OD600) was used to estimate the number of colony forming units of BCG in suspension during 3 hours measurements of clumping and 8 days measurements of BCG proliferation. BCG viability was evaluated using a substrate marker, MTT, which correlates with BCG density and metabolic activity. The anti-viral activity of IFN-alpha was determined in a cytopathic protection bioassay using the encephalomyocarditis virus/FS-4 cell system.nnnRESULTSnContinuous shaking of reconstituted BCG for 3 hours at 37C resulted in a marginal (11.3%) drop in OD600 which was minimally altered by inclusion of IFN-alpha at 2 million units (MU)/ml. (12.7% drop). Metabolic activity and growth rate of BCG alone or BCG with IFN-alpha were essentially identical. IFN-alphas antiviral activity was not affected by incubation with BCG.nnnCONCLUSIONSnThe inclusion of IFN-alpha into the usual BCG formulation for intravesical administration has no apparent effect on BCGs viability or tendency to form clumps in suspension. Similarly, the physical mixing of IFN-alpha with BCG does not impair its biological activity. Thus, both agents are pharmacologically compatible for future clinical studies involving combination intravesical therapy.

Mutated ras p21 as a target for cancer therapy in mouse transitional cell carcinoma.

PURPOSEnTo establish an experimental mouse model for bladder cancer immunotherapy using mutated ras as a target.nnnMATERIALS AND METHODSnA tumorigenic mouse bladder transitional cell carcinoma (TCC) line MB49 (C57BL/6 origin) was analyzed for its c-ras gene status by DNA cloning and sequencing. Aberrant expression of the ras gene was measured with Western blotting. 13-mer peptides corresponding to residues 5 to 17 of the ras protein were synthesized and tested for immunogenicity in syngeneic C57BL/6 mice. Induction of specific immune responses was evaluated by analyzing splenocyte activity in vitro and tumor suppression in vivo.nnnRESULTSnMB49 cells were found to contain a single amino acid substitution of serine for glycine at codon 12 in K-ras loci and an abundant amount of cellular mutated ras p21 protein. C57BL/6 mice immunized with the 13-mer serine-containing ras peptide exhibited mutation-specific immune responses in splenocyte proliferation, cytokine production and cytotoxicity. Specific antitumor immunity in the form of tumor growth delay in vivo was observed in mice immunized with the same mutant peptide followed by subcutaneous MB49 tumor challenge and was enhanced by the addition of low dose interleukin-12.nnnCONCLUSIONSnThe mouse bladder TCC line MB49 contains a serine mutation at codon 12 of its K-ras gene that is sufficient to induce mutation-specific immune responses in vitro and specific protective immunity to MB49 tumor in vivo. Mutated oncoproteins may be ideal targets for the development of specific immunotherapy regimens for bladder cancer immunotherapy.