Growth inhibition of HeLa cell by internalization of Mycobacterium bovis Bacillus Calmette-Guérin (BCG) Tokyo

Background  

Intravesical BCG immunotherapy is effective for preventing recurrence and progression in none muscle-invasive bladder cancer but the dosing schedule and duration of treatment remain empirical. The mechanisms by which intravesical BCG treatment mediates antitumor activity are currently poorly understood.     

Caseating granulomas on the glans penis as a complication of bacille calmette-guérin intravesical therapy

Intravesical instillation of bacille Calmette-Guérin (BCG) in patients with superficial bladder cancer or with carcinoma in situ of the bladder is a frequently used therapeutic option. As with other cancer therapies, BCG instillation has its share of side effects and complications. This case report highlights an uncommon complication.     

BCG-Mediated Bladder Cancer Immunotherapy: Identifying Determinants of Treatment Response Using a Calibrated Mathematical Model

Intravesical Bacillus Calmette Guérin (BCG) immunotherapy is considered the standard of care for treatment of non-muscle invasive bladder cancer; however the treatment parameters were established empirically. In order to evaluate potential optimization of clinical parameters of BCG induction therapy, we constructed and queried a new mathematical model. Specifically, we assessed the impact of (1) duration between resection and the first instillation; (2) BCG dose; (3) indwelling time; and (4) treatment interval of induction therapy – using cure rate as the primary endpoint. Based on available clinical and in vitro experimental data, we constructed and parameterized a stochastic mathematical model describing the interactions between BCG, the immune system, the bladder mucosa and tumor cells. The primary endpoint of the model was the probability of tumor extinction following BCG induction therapy in patients with high risk for tumor recurrence. We theoretically demonstrate that extending the duration between the resection and the first BCG instillation negatively influences treatment outcome. Simulations of higher BCG doses and longer indwelling times both improved the probability of tumor extinction. A remarkable finding was that an inter-instillation interval two times longer than the seven-day interval used in the current standard of care would substantially improve treatment outcome. We provide insight into relevant clinical questions using a novel mathematical model of BCG immunotherapy. Our model predicts an altered regimen that may decrease side effects of treatment while improving response to therapy.     

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.     

BCG strain S4-Jena: An early BCG strain is capable to reduce the proliferation of bladder cancer cells by induction of apoptosis

Background  

Intravesical immunotherapy with Mycobacterium bovis bacillus Calmette-Guérin has been established as the most effective adjuvant treatment for high risk non-muscle-invasive bladder cancer (NMIBC). We investigated the differences between the S4-Jena BCG strain and commercially available BCG strains. We tested the genotypic varieties between S4-Jena and other BCG strains and analysed the effect of the BCG strains TICE and S4-Jena on two bladder cancer cell lines.     

Immunotherapy for superficial bladder cancer

The treatment of superficial bladder cancer requires adjuvant therapies besides transurethral resection because of a high recurrence rate after this standard treatment alone. Current adjuvant therapies involve intravesical chemotherapy for patients at low and intermediate risk for recurrence and progression, and intravesical bacillus Calmette-Guérin for patients at intermediate and high risk. However, these adjuvant therapies fail in a significant number of patients, dictating the need for new and improved adjuvant treatment modalities for superficial bladder cancer. Immunotherapy aiming at the modulation of the immune system of the patient is a promising alternative adjuvant. This review discusses the current status of the clinical development of various immunotherapy approaches for superficial bladder cancer, including passive immunotherapy, immune stimulants, immunogene therapy and cancer vaccination.     

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.     

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.     

Localization and expression of inducible nitric oxide synthase in patients after BCG treatment for bladder cancer

Treatment with Bacillus Calmette Guerin (BCG) bladder instillations is an established treatment modality for superficial urinary bladder cancer and carcinoma in situ (CIS), but the anti-tumor mechanisms following BCG instillations remain largely unknown. Previous data show increased nitric oxide (NO) concentrations in the urinary bladder from patients treated with BCG suggesting that NO-formation may be involved in the BCG mediated effect. In the present study we evaluated 11 patients with urinary bladder cancer who had received BCG treatment and 11 tumor free control subjects. We performed immunohistochemistry, Western blot and real-time polymerase chain reaction (PCR) on bladder biopsies to establish inducible nitric oxide synthase (iNOS) protein levels and localization as well as iNOS mRNA expression. Endogenous NO formation in the bladder was also measured. In patients with bladder cancer who had received BCG treatment iNOS-like immunoreactivity was found in the urothelial cells but also in macrophages in the submucosa. Furthermore, endogenously formed NO was significantly increased (p<0.001) in the BCG treated patients and they had a ten-fold increase in mRNA expression for iNOS compared to healthy controls (p=0.003). In conclusion iNOS was found to be localized to the urothelium and macrophages underlying it. Our study also confirms elevated levels of endogenously formed NO and increased mRNA expression and protein levels for iNOS in patients with BCG treated bladder cancer. These data further support the notion that NO may be involved in the anti-tumor mechanism that BCG exerts on bladder cancer cells.     

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.     

肝癌免疫治疗的研究进展

肝细胞肝癌是全球发病率和死亡率最高的恶性肿瘤之一,发病率和死亡率呈逐年上升趋势。我国是肝癌大国,每年肝癌的死亡病例数位居全球第一。免疫治疗是继手术、化疗和放疗之后新兴的癌症治疗手段,其通过解除肿瘤微环境对免疫细胞的抑制作用并激活机体免疫功能,实现控制和杀伤肿瘤细胞。常用的免疫治疗的方法有免疫检查点治疗、过继免疫治疗和肿瘤疫苗治疗等。与传统治疗手段相比,免疫治疗因具有增强机体免疫功能、延缓肿瘤进展、延长患者生存时间等优点,逐渐成为基础和临床研究的热点。文中就免疫治疗在肝癌领域的研究进展作一综述。     

吡柔比星预防浅表性膀胱癌术后复发的疗效观察

目的：比较吡柔比星与吉西他滨膀胱内灌注预防浅表性膀胱癌术后复发的疗效。方法：40 例浅表性膀胱癌患者根据随机抽 签法分为治疗组与对照组各20 例,所有患者都采用经尿道膀胱肿瘤电切方法,对照组用吉西他滨,治疗组用吡柔比星进行膀胱 灌注,比较两组患者术后复发率的不同。结果：所有患者都完成治疗,随访1 年,治疗组的复发率为5.0 %,对照组为25.0 %,治疗 组的复发率明显低于对照组,对比差异明显,有统计学意义(P<0.05)。经过观察,治疗组的膀胱刺激症状、骨髓抑制、尿道狭窄等不 良反应总体发生率明显少于对照组,两者比较有统计学意义(P<0.05)。结论：相对于吉西他滨,吡柔比星膀胱内灌注预防浅表性膀 胱癌术后复发有很好的效果,不良反应少,在临床上需要根据患者的实际情况来选择不同的灌注药物。     

The Differential Expression of EphB2 and EphB4 Receptor Kinases in Normal Bladder and in Transitional Cell Carcinoma of the Bladder

Effective treatment of transitional cell carcinoma (TCC) of the bladder requires early diagnosis. Identifying novel molecular markers in TCC would guide the development of diagnostic and therapeutic targets. Ephrins mediate signals via tyrosine kinase activity that modulates diverse physiologic and developmental processes, and ephrins are increasingly implicated in carcinogenesis. The aim of our study was to examine the differential regulation of EphB4 and EphB2 in normal bladder and in TCC of the bladder in 40 patients undergoing radical cystectomy for curative intent. Immunostaining and Western blotting revealed that normal urothelium expresses EphB2 (20 of 24 cases, 83% of the time) not EphB4 (0 of 24 cases, 0%). In sharp contrast, TCC specimens show loss of EphB2 expression (0 of 34 cases, 0%) and gain of EphB4 expression (32 of 34, 94%). Furthermore, EphB4 signal strength statistically correlated with higher tumor stage, and trended toward the presence of carcinoma in situ (CIS). These results are confirmed by analysis of normal urothelial and tumor cell lines. EphB2 is not a survival factor in normal urothelium, while EphB4 is a survival factor in TCC. Treatment of bladder tumor xenograft with an EphB4 inhibitor sEphB4-HSA leads to 62% tumor regression and complete remission when combined with Bevacizumab. Furthermore, tissue analysis revealed that sEphB4-HSA led to increased apoptosis, decreased proliferation, and reduced vessel density, implicating direct tumor cell targeting as well as anti-angiogenesis effect. In summary loss of EphB2 and gain of EphB4 expression represents an inflection point in the development, growth and possibly progression of TCC. Therapeutic compounds targeting EphB4 have potential for diagnosing and treating TCC.     

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.     

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     

Follow-up investigations of bladder cancer patients by titration of natural and specific lymphocyte-mediated cytotoxicity

Summary A 44-hour incubation microcytotoxicity assay (MA) was used to titrate the lymphocyte-mediated cytotoxicity in 47 transitional cell carcinoma (TCC) bladder cancer patients and 65 clinical control patients. All titrations included three target cell lines: HU 456 (TCC), HU 609 (normal urothelium), and SAOS 2 (osteosarcoma). Tumor-specific cytotoxicity (TSC) was calculated as the difference between cytotoxicity to HU 456 and HU 609, and tumor type-specific cytotoxicity (TTSC) as the difference between cytotoxicity to HU 456 and SAOS 2. On the basis of TSC and TTSC values obtained before treatment TCC patients were divided into one group with high-grade specific cytotoxicity (HSC) and another with low-grade specific cytotoxicity (LSC). A prospective follow-up study of these patients revealed a significantly lower survival rate for patients with LSC compared with patients with HSC, even when the groups were corrected for differences in distributions according to clinical and histological tumor gradation. This indicates a growth-controlling function of the cellular immune reaction.Repeated cytotoxicity tests in a follow-up study of 26 TCC patients and one patient with a squamous cell carcinoma of the urinary bladder revealed a positive correlation between positive specific cytotoxicity and the presence of tumor tissue Gr 2–4. The reactivity vanished within 1 month after surgical removal of the tumor or at the end of radiotherapy. An increased cytotoxicity against HU 609, representing normal urothelium, was seen immediately following radiotherapy, and in a few cases after surgical treatment. Reapearance of elevated TSC and TTSC was noted during the months following radiotherapy. When the MA was considered as a diagnostic marker of tumor tissue during clinical control of patients with suspected TCC, 22% of positive reactions proved to be false-positive and 44% of negative reactions were false-negative. Thus, a negative result cannot be used to exclude recurrence, but a positive result may indicate the need for additional clinical examinations.Abbreviations NC natural cytotoxicity - TCC transitional cell carcinoma - TSC tumor-specific cytotoxicity - TTSC tumor type-specific cytotoxicity - MA microcytotoxicity assay - HSC high-grade specific cytotoxicity - LSC low-grade specific cytotoxicity - ICI integrated cytotoxicity index     

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.     

An Anti-Ubiquitin Antibody Response in Transitional Cell Carcinoma of the Urinary Bladder

Background

To use combinatorial epitope mapping (“fingerprinting”) of the antibody response to identify targets of the humoral immune response in patients with transitional cell carcinoma (TCC) of the bladder.

Methods

A combinatorial random peptide library was screened on the circulating pool of immunoglobulins purified from an index patient with a high risk TCC (pTa high grade plus carcinoma in situ) to identify corresponding target antigens. A patient cohort was investigated for antibody titers against ubiquitin.

Results

We selected, isolated, and validated an immunogenic peptide motif from ubiquitin as a dominant epitope of the humoral response. Patients with TCC had significantly higher antibody titers against ubiquitin than healthy donors (p<0.007), prostate cancer patients (p<0.0007), and all patients without TCC taken together (p<0.0001). Titers from superficial tumors were not significantly different from muscle invasive tumors (p = 0.0929). For antibody response against ubiquitin, sensitivity for detection of TCC was 0.44, specificity 0.96, positive predictive value 0.96 and negative predictive value 0.41. No significant titer changes were observed during the standard BCG induction immunotherapy.

Conclusions

This is the first report to demonstrate an anti-ubiquitin antibody response in patients with TCC. Although sensitivity of antibody production was low, a high specificity and positive predictive value make ubiquitin an interesting candidate for further diagnostic and possibly immune modulating studies.     

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.