Detection of human melanoma antigens in cell-free supernatants.

Common human melanoma membrane antigens have been demonstrated by immunofluorescent microscopy in cultured melanoma cells using antisera raised in humans by autoimmunization of patients with their own irradiated tumor cells mixed with adjuvant BCG. The melanoma antigens will migrate, cap, and become extruded on the cell surface when they are treated with the postimmune anti-melanoma sera. Some of these antigens are spontaneously shed into the culture media even without added antibody. Those shed antigens are stable to dialysis and lyophilization and may be isolated from a Sephadex G-200 solumn along with proteins of molecular weight 80,000–150,000 by membrane fluorescence inhibition and micro-complement fixation. Further purification of such antigens may facilitate the development of radio or enzymoimmunoassay which will permit sensitive measurement of antigens in melanoma patients.     

What is the optimal regimen for BCG intravesical therapy? Are six weekly instillations necessary?

OBJECTIVE: For more than 20 years, BCG intravesical therapy schedule has included 6 weekly instillations. Very few studies have, however, analyzed the rationale of this regimen. We previously demonstrated that intravesical BCG induced an increased peripheral immune response against mycobacterial antigens as compared to pretreatment values. In the present work, we have studied the weekly evolution of this immune response induced by intravesical BCG instillations. MATERIALS AND METHODS: The evolution of the lymphoproliferative response of peripheral blood mononuclear cells against BCG culture filtrate (CF), tuberculin (PPD) and BCG extract (EXT) was tested before, every week during the BCG instillations and at 3 and 6 months follow-up in 9 patients with superficial bladder cancer treated with 6 weekly BCG instillations. Lymphoproliferation was measured by means of a tritiated thymidine incorporation test. RESULTS: A significant increase in the lymphoproliferative response against PPD, CF and EXT was observed in 9, 8 and 7 of the 9 patients, respectively, as compared to pre-BCG values. The maximal lymphoproliferation was achieved after 4 instillations in 4/5 patients initially reactive against mycobacterial antigens whereas 2 of 4 initially nonreactive patients required 6 instillations. At 6 months' follow-up, lymphoproliferation against BCG and the other mycobacterial antigens returned to pre-BCG values in all patients. In 3 patients who received additional instillations because of tumor recurrence within 1 year of follow-up, the maximum immune response was observed already after 2 instillations. CONCLUSION: In most patients, the maximal peripheral immune response is already observed after 4 weekly instillations. However, patients not previously immunized against mycobacterial antigens may require 6 weekly instillations to achieve a maximum stimulation level. Our data support the need to further evaluate the role of this status before starting BCG instillations. It could be of interest to study whether 6 BCG instillations are really necessary in patients previously immune against mycobacterial antigens.     

BCG Immunotherapy of Malignant Melanoma: Summary of a Seven-year Experience

Over the past 7 years, 151 patients with malignant melanoma have been treated with BCG immunotherapy alone or as an adjunct to surgical therapy. Direct injection of metastatic melanoma lesions limited to skin resulted in 90% regression of injected lesions and 17% regression of uninjected lesions in immunocompetent patients. Approximately 25% of these patients remained free of disease for 1 to 6 years. Direct injections of BCG into nodules of patients with subcutaneous or visceral metastases resulted in a lower incidence of local control and no long term survivors. Attempts to improve the results of immunotherapy in these patients by palliative surgical resection of large metastatic lesions to lower tumor burden followed by BCG immunotherapy significantly improved the results although many patients still developed recurrent disease. Early results of a clinical trial combining BCG immunotherapy with regional lymphadenectomy in patients with melanoma metastatic to lymph nodes have been encouraging and promising. Further controlled clinical trials are necessary to elucidate the role of BCG in immunotherapy. However, since BCG is but one of a number of potential immunologic adjuvants, even more effective immunotherapy will be possible as further knowledge of the interactions of cellular and humoral immunity is acquired.     

Effect of bacillus Calmette-Guerin immunotherapy on tumor antigen-induced lymphocyte-stimulated protein synthesis in melanoma patients.

Changes in in vitro lymphocyte-stimulation protein synthesis (SPS) of 40 melanoma patients following incubation with 3M KCl extracts of allogenic melanoma, lung carcinoma, and sarcoma antigens and phytohemagglutinin (PHA) were quantitated by measuring H-3-leucine uptake. One of eleven "untreated" melanoma patients stimulated significantly to the melanoma antigen. However, this lymphocyte response was not significantly different from that of the normal subjects. Patients who received systemic bacillus Calmette-Guerin (BCG) by the tine technique for 3 months and for 6 months had significant increase in lymphocyte protein synthesis following incubation with melanoma antigen. There were no significant differences in PHA responses between the "untreated" melanoma patients and the BCG-treated group. Testing of serial lymphocyte samples from nine melanoma patients before treatment and at monthly intervals thereafter confirmed these observations. Furthermore, no change in serial complement-fixing antibody titers to melanoma antigen was noted in the BCG-treated patients. These results demonstrated that in vitro lymphocyte responses to melanoma antigen may be augmented by BCG therapy.     

Local immune responses after intravesical BCG treatment for carcinoma in situ

The lymphoid cellular infiltrates in the bladder biopsies from patients with carcinoma in situ before and after BCG intravesical therapy have been determined and characterised. This has been achieved using a panel of monoclonal antibody probes in an indirect immunoperoxidase technique. These studies have revealed the predominance of T-cells of the helper/inducer phenotype (T4+), beta-lymphocytes (B1+) and macrophages (Leu M3+, 3.9+) in bladder biopsies after BCG therapy. HLA.DR antigens were also expressed on the lymphoid cells infiltrate as well as the urothelial cells. These results suggest that the components of an active immune response were present and enhanced in the bladder wall after BCG therapy.     

Bacillus Calmette-Guerin (BCG): Its fight against pathogens and cancer

Bacillus Calmette-Guerin (BCG) is the only FDA approved first line therapy for patients with nonmuscle invasive bladder cancer. Since the turn of the 20th century BCG has been used as a vaccine for protection against Mycobacterium tuberculosis (Mtb) and has also been found to have protection against nontuberculosis related pathogens. Recently the role of “trained immunity” has been identified as a possible mechanism for BCG vaccine-mediated immunity to Mtb. Similarly, BCG has been used as an immunotherapy for bladder cancer for more than 40 years, and the underlying mechanisms for BCG-mediated anti-tumor activity is poorly characterized. Several studies have shown that multiple immune pathways contribute to the immune response, and efficacy of intravesicle BCG as a cancer therapy. It is vital that we integrate our understanding of BCG as a vaccine and as a cancer therapeutic to facilitate design of future studies in order to maximize the immunotherapeutic potential of BCG. In this review we will outline the role of BCG as a vaccine, the known immune pathways that are activated by intravesical BCG and outline a potential clinical study integrating BCG vaccination prior to intravesicle instillation of BCG.     

Active Antigen-specific Immunotherapy of Melanoma: from Basic Science to Clinical Investigation

Advanced-stage melanoma here dismal prognosis, and novel therapeutic approaches are urgently required. The possibility of taking advantage of the immune response of patients for its treatment has been an appealing concept for almost a century. Only during the last decade, however, has the molecular identification of tumor-associated antigens (TAAs) offered the possibility of vaccinating patients (e.g., active induction of TAA-specific immune responses). Active antigen-specific immunotherapy (AASIT) is currently being investigated in a number of clinical centers as a treatment option for advanced-stage melanoma. A large number of melanoma TAAs have been molecularly characterized and are being used in vaccination trials in various molecular forms and according to various immunization protocols. Here we provide a short overview on melanoma TAAs, the technologies currently in use to induce specific cytotoxic T-lymphocyte (CTL) responses in vivo, and their monitoring. We also propose a tentative AASIT agenda for the next few years, aiming at improving the capacity to induce and monitor TAA-specific immune responses and to verify their clinical effectiveness.This work was partially funded by grants from the Swiss Bridge Foundation and by the Swiss Science Foundation.     

The survival of patients with malignant melanoma receiving BCG with or without chemotherapy.

The effects of treatment by immunotherapy and chemoimmunotherapy were assessed in 31 non-randomized patients with melanoma; 16 received only BCG and 15 BCG and chemotherapy. For advanced cases, the times of survival for the two treatment groups did not differ significantly, nor did there appear to be any improvement in survival over that recorded for the natural course of the disease. The pretreatment immune status as judged by hyporeactivity or normal reactivity to skin tests for delayed type hypersensitivity did not appear to influence survival. This study, together with other reports, does not support the use of immunotherapy in advanced melanoma. Immunotherapy for early melanoma is still to be assessed.     

THE SURVIVAL OF PATIENTS WITH MALIGANT MELANOMA RECEIVING BCG WITH OR WITHOUT CHEMOTHERAPY

The effects of treatment by immunotherapy and chemoimmunotherapy were assessed in 31 non-randomized patients with melanoma; 16 received only BCG and 15 BCG and chemotherapy. For advanced cases, the times of survival for the two treatment groups did not differ significantly, nor did there appear to be any improvement in survival over that recorded for the natural course of the disease. The pretreatment immune status as judged by hyporeactivity or normal reactivity to skin tests for delayed type hypersensitivity did not appear to influence survival. This study, together with other reports, does not support the use of immunotherapy in advanced melanoma. lmmunotherapy for early melanoma is still to be assessed.     

CURRENT STATUS AND FUTURE PROSPECTS FOR ADJUVANT THERAPY OF MELANOMA

Advances in the treatment of melanoma have resulted mainly from improved surgical management of the primary tumour assisted by a greater appreciation of major prognostic factors in the natural history of the disease. Further improvement in the treatment of melanoma will depend largely on introduction of methods to prevent recurrence of the disease. The present review discusses criteria for selection of patients with a high risk of recurrent disease and the adjuvant treatment that has been used in past studies to prevent recurrences. With few exceptions various regimens of chemotherapy, non-specific immunotherapy with bacterial products or combinations of these treatments have not increased disease free or survival periods. Immunotherapy with various sources of melanoma antigens or with viral lysates of melanoma cells have produced encouraging results in uncontrolled studies and require further evaluation. Several advances appear to provide scope for new initiatives in immunotherapy. These include an appreciation of the role of suppressor cells in regulation of immune responses against tumour cells and possible methods to inhibit their activity. A second is the definition of various lymphokines involved in generation of immune responses (particularly interleukin 2) and development of in vitro methods for large scale production of these factors. Thirdly, methods are becoming available to define the heterogeneity of tumour cells in terms of cell surface antigens or their release of soluble factors which may help select treatments appropriate to each patient.     

SUPERFICIAL BLADDER TUMORS AND INCREASED REACTIVITY AGAINST MYCOBACTERIAL ANTIGENS BEFORE BACILLUS CALMETTE-GUERIN THERAPY

Purpose

The precise mechanism of action of bacillus Calmette-Guerin (BCG) in bladder cancer treatment remains poorly understood. Whether bladder tumor cells are destroyed by nonspecific mechanisms or targeted by specifically activated lymphocytes recognizing cognate antigens is unclear. To investigate a possible cross-reactivity between BCG and bladder cell tumors, we tested before BCG treatment the lymphoproliferation of peripheral blood lymphocytes against several mycobacterial antigens, including the secreted fibronectin binding antigen 85 complex from BCG (AG 85) in patients with superficial bladder tumors compared to control matched patients.

Materials and Methods

Using a whole blood assay, T cell response against purified protein derivative, BCG extract, whole BCG, purified AG 85, and the nonspecific mitogens pokeweed and phytohemagglutinin was investigated in 79 patients with superficial bladder tumors before BCG and in 39 control subjects without malignancy matched for age and sex. Neither group had a history of tuberculosis. Lymphoproliferation was measured with a tritiated thymidine uptake assay on day 7 of culture.

Results

Of the 79 patients with superficial transitional cell carcinoma, a significant lymphoproliferative response before BCG against PPD, BCG extract, whole BCG and AG 85 was observed in 65 (82.2%), 67 (84.81%), 30 (37.97%) and 49 (62.02%) patients, respectively. Of the 39 controls only 26 (64.1%), 23 (58.9%), 3 (7.7%) and 3 (7.7%) patients, respectively, had a significant lymphoproliferation against PPD, BCG extract, BCG and AG 85 (p >0.05, p = 0.004, p = 0.00001 and p = 0.00001, respectively). In terms of lymphoproliferative levels, patients with superficial transitional cell carcinoma also showed a significantly higher response against PPD (p = 0.000012), BCG extract (p = 0.000001), AG 85 (p = 0.000001), whole BCG (p = 0.00001) and pokeweed (p = 0.01) than controls but not against phytohemagglutinin.

Conclusions

Patients with superficial transitional cell carcinoma demonstrate an increased lymphoproliferation against mycobacterial antigens before BCG compared to control subjects. Although a nonspecific activation of the immune system cannot be excluded at this stage, our data may suggest the possible existence of bladder cancer antigens cross-reactive with mycobacterial antigens responsible for boosting precursor cells witnessing previous contacts with mycobacteria. The implication of these findings in the antitumoral mechanism of action of BCG are under investigation.     

Lung cancer-associated tumor antigens and the present status of immunotherapy against non-small-cell lung cancer

Despite recent advances in surgery, irradiation, and chemotherapy, the prognosis of patients with lung cancer is still poor. Therefore, the development and application of new therapeutic strategies are essential for improving the prognosis of this disease. Significant progress in our understanding of tumor immunology and molecular biology has allowed us to identify the tumor-associated antigens recognized by cytotoxic T lymphocytes. Immune responses and tumor-associated antigens against not only malignant melanoma but also lung cancer have been elucidated at the molecular level. In a theoretical sense, tumor eradication is considered possible through antigen-based immunotherapy against such diseases. However, many clinical trials of cancer vaccination with defined tumor antigens have resulted in objective clinical responses in only a small number of patients. Tumor escape mechanisms from host immune surveillance remain a major obstacle for cancer immunotherapy. A better understanding of the immune escape mechanisms employed by tumor cells is necessary before we can develop a more effective immunotherapeutic approach to lung cancer. We review recent studies regarding the identification of tumor antigens in lung cancer, tumor immune escape mechanisms, and clinical vaccine trials in lung cancer.     

A clinical trial of BCG immunotherapy as an adjunct to surgery in the treatment of primary malignant melanoma.

A preliminary analysis of a controlled trial of BCG immunotherapy as an adjunct to surgery in the treatment of primary malignant melanoma has been carried out. The length of follow-up varied from 5 years to 6 months. No obvious benefit from BCG immunotherapy has been found so far. On the other hand the treatment is painful with an appreciable morbidity. Skin tests for delayed hypersensitivity have shown no recognisable differences in patients treated with surgery and those who also had BCG, or in the pattern of responses in those who developed recurrences and those who did not. In view of these early findings trial entry has been closed.     

Recombinant BCG therapy suppresses melanoma tumor growth

Background: Melanoma is the fastest rising cancer in the United States. Bacillus Calmette-Guerin (BCG) has been genetically engineered to actively express and secrete the cytokine interleukin-2 (IL-2). Both BCG and IL-2 have known potent antitumor and immunomodulatory properties. Methods: This recombinant BCG (rBCG 3A) has been tested as an intratumoral injection and a vaccine therapy in conjunction with irradiated tumor cells against melanoma in the murine B16 melanoma model. Results: The transfection process did not adversely after the function of the wild-type (WT) BCG. rBCG 3A and WT BCG are equally effective intratumoral and vaccine therapies against melanoma when compared with normal saline control groups. Tumor burdens were significantly smaller (p</0.01 and 0.05) for the treatment groups for both intratumoral and vaccine administration of therapy. Immunization with rBCG 3A and WT BCG 14 days before a B16 challenge resulted in an ∼45% smaller tumor burden when compared with controls. Conclusions: Novel therapies based on the immunogenic properties of melanoma combined with molecular technologies may offer promise for an effective and safe treatment of melanoma. Results of this study were presented at the 47th Annual Cancer Symposium of The Society of Surgical Oncology, Houston, Texas, March 20, 1994.     

Follow-up of the effect of BCG in bladder tumour patients

The intravesical BCG effect in 38 patients with superficial bladder tumour after TUR was followed by the lymphocyte transformation test (LTT) and the staphylococcus phagocytosis test of in vitro washed leukocytes. The results have confirmed the immunostimulating and hence anti-tumour effect of intravesical BCG. The beginning and the duration of the stimulation effect were defined and the necessity of maintenance treatment was verified. Authors consider monitoring of the cellular immune response suitable for the continuous follow-up of the BCG effect. Comparing the tolerable side-effects with their favourable therapeutic results, BCG is considered to be suitable for the prevention of recurrences in treating superficial bladder tumours.     

Durability of complete responses in patients with metastatic cancer treated with high-dose interleukin-2: identification of the antigens mediating response.

OBJECTIVE: To determine the durability of complete responses in patients with metastatic melanoma or renal cancer treated with high-dose bolus interleukin-2 (IL-2) as well as the factors associated with the development of a complete response and the antigens mediating clinical responses. METHODS: A consecutive series of 409 patients with either metastatic melanoma or renal cancer who were treated with high-dose bolus IL-2 in the Surgery Branch, National Cancer Institute, between September 1985 and November 1996 have been analyzed with a median potential follow-up of 7.1 years. All patients were treated with 720,000 IU/kg administered by 15-minute intravenous infusions every 8 hours for up to 5 days as clinically tolerated per cycle. Two cycles constituted a treatment course. Tumor-infiltrating lymphocytes (TIL) from melanoma patients were used to clone the genes encoding the tumor antigens responsible for clinical responsiveness. RESULTS: Thirty-three of 409 (8.1%) patients treated with high-dose bolus IL-2 achieved a complete response and 37 (9%) achieved a partial response. Complete regression was seen in 6.6% and 9.3% of patients with metastatic melanoma and renal cancer, respectively. Twenty-seven of these 33 completely responding patients (82%) remain in ongoing continuous complete response from 39 to more than 148 months from the onset of treatment. Tumor regressions were seen at virtually all organ sites. The absence of prior treatment with immunotherapy, the total dose of IL-2 administered, and the maximal rebound lymphocytosis after cessation of IL-2 correlated with achieving a complete response. Expression cloning techniques have identified a series of tumor antigens that are recognized by TIL grown from resected melanomas. These antigens are mainly melanoma/ melanocyte differentiation antigens, although mutated intracellular proteins can also serve as antigens. CONCLUSIONS: Treatment with high-dose bolus IL-2 mediates complete cancer regression in approximately 8% of patients with metastatic renal cancer and melanoma. The great majority of these patients will enter durable complete regressions and appear to be cured of their metastatic cancer. Thus, immunotherapy with high-dose bolus IL-2 should be considered as initial therapy for appropriately selected patients with metastatic melanoma and renal cell cancer. Identification of the tumor antigens mediating clinical response is opening new therapeutic possibilities for cancer treatment.     

Requirement of a thymus dependent immune response for BCG-mediated antitumor activity

Surgical adjuvant intravesical bacille Calmette-Guerin (BCG) therapy is an effective method of treating superficial transitional cell carcinoma of the bladder. The role of the immune response in the antitumor activity of intravesical BCG is not known. We investigated the requirement of a thymus-dependent immune response for the inhibition of the growth of the intravesically implanted mouse bladder tumor, MBT-2. Intravesical BCG had no antitumor activity when administered to athymic nude mice bearing MBT-2 tumors. In two experiments tumor outgrowth in control and BCG-treated mice was identical. Adoptive transfer of BCG sensitized splenocytes (one spleen equivalent per mouse injected intravenously immediately prior to the first BCG treatment) syngeneic to the MBT-2 tumor transferred delayed hypersensitivity reactivity to BCG antigens and restored the antitumor activity of intravesical BCG. In two separate experiments mice receiving splenocytes plus BCG had 0 and 20% tumor outgrowth compared with 100% in control mice (p less than .02 and p less than .05, respectively). These results demonstrate that the antitumor activity of intravesical BCG therapy requires a thymus-dependent immune response.     

Preliminary results of a randomized trial of adjuvant immunotherapy in patients with malignant melanoma who have lymph node metastases.

This study evaluates the effect of adjuvant immunotherapy with BCG alone, or combined with melanoma cell vaccine, on the recurrence and survival rates of patients with metastatic melanoma in the regional lymph nodes who were treated by lymphadenectomy. Patients were prospectively randomized and stratified on the basis of age, sex, site of primary tumour, and clinical estimate of the regional nodes. During the past four years, 134 patients were entered into this trial, and to date, the incidence of recurrence among the two arms mentioned and the control arm is not significantly different; however, patients receiving BCG alone had longer survival than those in either the tumour cell vaccine or control groups. The improved survival in the BCG-only group was found to be due to the fact that patients survived longer with their recurrent disease than the patients in the other two groups. At the present time, these differences do not appear to be significant enough to justify routine adjuvant immunotherapy in patients with melanoma metastatic to regional nodes. Longer follow-up will be necessary to evaluate the role of adjuvant immunotherapy of Stage II melanoma.     

ENDOLYMPHATIC ISOTOPE AND BCG IN THE MANAGEMENT OF MALIGNANT MELANOMA

Endolymphatic isotope therapy had such promising early clinical results that the M.R.C. (Medical Research Council) U.K. set up a clinical trial in 1966. This was to compare the effect of endolymphatic isotope therapy with the results of standard methods in the treatment of lower limb malignant melanoma. The interim report had three groups for analysis; Standard Methods (S); Endolymphatic Satisfactory (ES); and Endolymphatic Unsatisfactory (EU). This third group was a subdivision, as a significant number of patients did not have the correct endolymphatic treatment. The five-year survival figures expressed as actuarial percentages were ES=78.8%; S=82.3%; and EU = 57.3%. Lymph node recurrence showed a significant difference: ES=2.3%; EU=12%; and S=19%. The conclusions were that endolymphatic isotope therapy was justified in specialized centres where good results could be obtained. Further animal experiments using the VX2 tumour in rabbits indicated that BCG given intracutaneously or intravenously had no therapeutic effect, whereas when applied by intralymphatic injection BCG was successful in treating lymph node metastases. Nineteen patients with poor-prognosis malignant melanoma have received endolymphatic BCG. The clinical results are recorded in this paper and are sufficiently encouraging to warrant its continued use.     

Endolymphatic isotope and BCG in the management of malignant melanoma.

Endolymphatic isotope therapy had such promising early clinical results that the M.R.C. (Medical Research Council) U.K. set up a clinical trial in 1966. This was to compare the effect of endolymphatic isotope therapy with the results of standard methods in the treatment of lower limb malignant melanoma. The interim report had three groups for analysis: Standard Methods (S); Endolymphatic Satisfactory (ES); and Endolymphatic Unsatisfactory (EU). This third group was a subdivision, as a significant number of patients did not have the correct endolymphatic treatment. The five-year survival figures expressed as actuarial percentages were ES=78.8%; S=82.3%; and EU=57.3%. Lymph node recurrence showed a significant difference: ES=2.3%; EU=12%; and S=19%. The conclusions were that endolymphatic isotope therapy was justified in specialized centres where good results could be obtained. Further animal experiments using the VX2 tumour in rabbits indicated that BCG given intracutaneously or intravenously had no therapeutic effect, whereas when applied by intralymphatic injection BCG was successful in treating lymph node metastases. Nineteen patients with poor-prognosis malignant melanoma have received endolymphatic BCG. The clinical results are recorded in this paper and are sufficiently encouraging to warrant its continued use.