Immunotherapy of breast cancer,malignant melanoma,and acute leukemia with BCG: Prolongation of disease free interval and survival

Summary Results of immunotherapy with BCG in patients with malignant melanoma, breast cancer, and acute leukemia are described. The first study demonstrated that high doses of living BCG organisms (6×10⁸ viable units) delivered by scarification in the upper arms and legs prolonged the disease-free interval and survival of 52 malignant melanoma patients with regional lymph node metastases compared to 218 comparable surgical control patients. Patients with trunk and extremity, but not head and neck melanoma, benefited from BCG, suggesting the importance of the delivery of BCG into the tumor-involved lymphatics.The second study evaluated the therapeutic efficacy of living BCG organisms by scarification in a group of adult acute leukemia patients after the cessation of chemotherapy. Thirty-seven patients had been in remission on intermittent chemotherapy for 12–24 months. Following late intensive consolidation chemotherapy, 7 consecutive patients received no further therapy and then 30 consecutive patients received BCG. Patients maintained on BCG have had a prolonged disease-free interval compared to those given on no further therapy (P=0.07) or compared to a group of similar patients maintained on chemotherapy alone (P=0.001). Similarly, the survival has been improved for patients maintained on BCG compared to those left unmaintained (P=0.009), or those maintained on chemotherapy (P=0.001).The principles of intermittent chemotherapy combined with BCG immunotherapy, first developed in patients with disseminated melanoma and acute myelogenous leukemia, have been confirmed in a series of patients with disseminated breast cancer. Forty-five patients treated with a combination of 5-FU, adriamycin, and cyclophosphamide (FAC) plus BCG by scarification showed prolongation of remission as well as survival compared to a comparable group of 44 patients treated with FAC chemotherapy without immunotherapy. Thus, 23/44 patients treated with FAC have died (median=14 months) compared to only 5/45 patients on FAC-BCG (median=12⁺ months), P=0.005. The limitations of BCG immunotherapy as well as speculations for future developments of immunotherapy are discussed.This work was supported by Contract No1-CB 33888 from the National Institutes of Health, Public Health Service, Bethesda, Maryland 20014. Drs. Gutterman and Mavligit are the recipients of Career Development Awards (Ca 71007-02 and CA 00130-01, respectively) from the National Institutes of Health, Education, and Welfare, Bethesda, Maryland 20014.     

Various uses of BCG and allogeneic acute leukemia cells to treat patients with acute myelogenous leukemia

Summary Ninety-six remission patients with acute myelogenous leukemia have been treated with various forms of immunotherapy and chemotherapy in three distinct studies and the clinical outcome of these patients has been reported. In the first study 22 patients were maintained on chemotherapy alone and 28 patients were given the same chemotherapy and additional immunotherapy consisting of BCG and irradiated allogeneic AML cells given at separate sites weekly. It was found that there was a significant increase in survival time of the patients who received immunotherapy (median 510 days) compared with the chemotherapy alone patients (270 days). The p value for this was 0.03. The reason for this prolongation of survival was mainly due to a markedly increased survival time of immunotherapy patients after they relapsed when compared with the chemotherapy patients (165 days compared with 75 days median, p equal to 0.0005). In the second sequential study 24 patients were given immunotherapy alone consisting of irradiated allogeneic AML cells and BCG given at separate sites, and this was compared with unirradiated allogeneic cells and BCG given to 22 patients. There was no difference in the remission length or survival between these two groups. In the third study 13 patients received irradiated cells and BCG as in Study 1 and a further 11 patients received the same immunotherapy but also received a mixture of cells and BCG given during the first three months. There was no difference in the remission and survival of these two groups. The significance of these results is discussed.     

A controlled trial of BCG immunotherapy in bronchogenic carcinoma treated by surgical resection

Summary Ninety-two patients with bronchogenic carcinoma who were treated by surgical resection of the tumour were subsequently given immunotherapy with BCG (Glaxo). The study was strictly randomised into three groups. Twenty-nine patients received multipuncture BCG (50–250×10⁶ viable units) and 26 patients intradermal BCG (0.4–0.9×10⁶ viable units) treatment being given at 1, 2, 5, 9, 13 and 26 weeks after operation and every 26 weeks thereafter. Thirty-seven control patients did not receive BCG. The patients have been observed for 15–33 months. There was no significant difference in survival between the control group and the two immunotherapy groups or between the two immunotherapy groups. The tumour cell type and presence of mediastinal nodes significantly influenced overall survival but not the response to BCG immunotherapy. The possible reasons for the failure of BCG to prolong survival in this study are discussed.     

Evaluation of time and dose in treating mammary adenocarcinoma with immunostimulants

Summary BCG, C. parvum, and reovirus were used as immunostimulants in treating murine mammary adenocarcinoma (A-10) after tumor burden had been minimized with BCNU. Immunostimulants were administered at different times with respect to chemotherapy. Different doses were used to determine the optimal response as measured by survival. BCG induced the best response when 6.67×10⁵ organisms were given 2 days after chemotherapy. The optimal response with C. parvum was observed after a dose of 0.35 mg was given 1 or 2 days after chemotherapy. Similarly, reovirus produced the best response when 10¹⁰ plaque-forming units were given 2 days after chemotherapy. These data are consistent with previous findings and support the notion that immunostimulants require an appropriate lymphoid substrate in order to induce an adequate anti-tumor response.Tge abbreviations used are: BCNU: 1,3-bis-(2-chloroethyl)-1-nitrosourea Saline: 0.9% NaCl solution; BCG: Bacillus Calmette-Guerin; C. parvum: Corynebacterium parvum; pfu: plaque-forming unitsThis study was supported, in part, by Contract No. N01-CB-43864 and Grant No. CA 14460 from The National Cancer Institute     

Antimicrobial resistance in bacteria from breeding dogs housed in kennels with differing neonatal mortality and use of antibiotics

This work examines the antimicrobial resistance of potentially pathogenic bacteria (Staphylococcus pseudintermedius, Streptococcus canis, Escherichia coli) found in the vaginal tract in prepartum mammary secretions and postpartum milk of bitches housed in breeding kennels (N = 20; 92 bitches). The kennels were divided into three categories: no routine antimicrobial administration around parturition (category 1); routine administration of one antibiotic around parturition (category 2); routine administration of multiple antimicrobials around parturition (category 3). Bacteriological cultures and antibiotic susceptibility tests were performed on vaginal specimens, prepartum mammary secretions, and postpartum milk. Stillbirths and neonatal deaths were recorded for each whelping and analyzed as “within-litter stillbirths” and “within-litter neonatal deaths” according to kennel category, by Pearson χ² test and the Kruskal-Wallis nonparametric test, respectively. The frequency of isolation and antimicrobial resistance of bacteria were analyzed according to kennel category by Pearson χ² test. Kennel category was not significantly associated with differing numbers of stillbirths or neonatal death events, nor was the frequency of isolation of potentially pathogenic bacteria in the three kennel categories significantly different. Kennel category 3 had a significantly higher frequency of isolation of multiresistant gram-positive bacterial strains. Our results show that intense administration of antibiotics to breeding bitches does not effectively reduce neonatal mortality; on the contrary, it induces multiresistance in potentially pathogenic bacteria. Breeders and veterinarians should be aware of the risk of selecting pathogenic bacteria by uncontrolled treatment in prepartum bitches.     

Canine osteosarcoma failure of intravenous or intralesional BCG as adjuvant immunotherapy

Summary Dogs with spontaneous osteosarcoma of an extremity were entered into two consecutive trials of adjuvant immunotherapy with BCG. In the first trial, 30 dogs underwent amputation followed by intravenous BCG, 0.4–1.6×10⁸ viable organisms, on the day of amputation, 1 and 3 weeks later and then monthly for 1 year. In the second trial, 2–8×10⁸ viable BCG organisms or 6 mg BCG cell walls in oil were injected intralesionally 10–26 (median=17) days before amputation. Neither time to development of metastatic disease nor survival was prolonged by either immunotherapy protocol.Dr. Deeg is a Fellow of the Leukemia Society of America     

Active specific immunotherapy of residual micrometastasis

Summary A vaccine of Bacillus Calmette-Guérin (BCG) admixed with tumor cells induced systemic immunity and had a therapeutic effect on subclinical, disseminated micrometastasis. Inbred strain-2 guinea-pigs given IV injections of 5×10³ to 10⁶ syngeneic L10 hepatocarcinoma cells were vaccinated after metastatic foci were established in the lung parenchyma. The purpose of this study was to establish the variables that can be manipulated to assure optimal immunotherapy while minimizing deleterious side effects of the BCG. In the present study we examined the variables of source, dose, and ratio of BCG to tumor cells. Four BCG sources (lyophilized Tice and Connaught; fresh-frozen Phipps and Tice) were compared. No significant differences among these BCG preparations could be detected with respect to adjuvant potential when they were admixed with attenuated tumor cells in a vaccine. The dose study clearly demonstrated that a BCG dose dependency exists with relation to induction of effective cell-mediated immunity or survival from disseminated micrometastatic disease. Furthermore, evaluations of dose versus ratio of BCG to tumor cells also supported a BCG dose dependency, with the lowest effective BCG dose being directly influenced by tumor burden of the host. Cutaneous reactivity and hypersensitivity of the primary and secondary immunization sites of tumor-bearing animals treated with effective and ineffective vaccines supported the direct association of reaction to BCG and specific tumor immunity. However, when an in vitro leukocyte migration inhibition assay was used, the degree of reactivity to BCG could not be exploited as a quantitative, diagnostic monitor of effective systemic tumor immunity.     

BCG in the immunotherapy of malignant lymphomas

Summary The effectiveness of a nonspecific immunostimulation in related human or animal diseases incited us to do a study of nonspecific immunotherapy by BCG in Hodgkin's disease, and then in other malignant lymphomas. Seventy patients, each one fulfilling at least 2 criteria of poor prognosis, were initially put in complete remission by a combination of radio-chemotherapy, followed by a reinforcing chemotherapy. These patients were then randomized into two groups. The first group received no further treatment; the second received BCG in weekly cutaneous scarifications. Eight patients were excluded from the study. The rate of relapses is significantly lower in the treated group. The results are discussed. Other therapeutic studies are necessary to fix the indications and modalities of this immunotherapy.Communication to the Medical Oncology Society, Nice, December 7, 1976     

Equine sarcoid: BCG immunotherapy compared to cryosurgery in a prospective randomised clinical trial

Summary A total of 30 horses with single or multiple sarcoid tumors of the skin were randomly divided into three treatment groups: (i) cryosurgical treatment, (ii) intralesional immunotherapy with a live BCG vaccine, (iii) intralesional immunotherapy with a BCG cell wall preparation. Complete tumour regression was obtained in all 10 crysurgically treated horses, in 6 of 10 live BCG treated horses, and in 7 of 10 BCG cell wall treated horses. One live BCG and 2 BCG cell wall treated horses showed partial tumour regression of more than 50% of the tumour area. Eleven horses with sarcoid tumours were not eligible for random allocation in the trial because unfavourable site or size of the tumour precluded cryosurgical treatment. These animals were treated with BCG cell wall vaccine except for 1 animal, which was treated with live BCG. In 4 cases this treatment was combined with cytoreductive surgery of the tumour. In this prognostically unfavourable group 8 animals showed complete tumour regression and 3 animals did not respond.Regression after BCG immunotherapy appeared to correlate with size (larger tumours worse response) and localization of the sarcoid (less favourable results in the limb), and increase in peripheral blood leucocytes after the first injection. Horses with a positive delayed type hypersensitivity reaction to PPD before the start of treatment showed a tendency to more favourable prognosis than PPD negative horses. No correlation was present between regression and single or multiple presence of sarcoids, increase in body temperature after injection of BCG and the formation of specific antibodies to BCG. None of the cured animals have shown tumour recurrence 3 to 40 months following treatment.Animals were maintained under the guidelines prescribed by the Faculty of Veterinary Medicine, State University Utrecht, The NetherlandsGrant recipient of the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation)     

Adjuvant immunotherapy in acute nonlymphocytic leukemia

Summary Of 112 patients (maximum age 70 years) with acute nonlymphocytic leukemia, 62 (55%) went into remission on an induction therapy of cytosine arabinoside and daunorubicin. 20 patients were randomized for maintenance treatment consisting of chemotherapy only and 22 patients for combined chemo-immunotherapy. The chemotherapy consisted in 5-day courses of daunorubicin and cytosine arabinoside and of thioguanine and cytosine arabinoside, alternating every month. The chemo-immunotherapy group also received weekly intracutaneous injections of 10⁹ allogeneic nonirradiated leukemic myeloblasts and 10⁶ BCG organisms (Glaxo) by Heaf gun.The median duration of the first remission was 164 days for the chemotherapy group and 464 days for the chemo-immunotherapy group. The corresponding median times of survival were 344 days for the first group and 734 days for the second group. The difference concerning median duration of survival is statistically significant. Thus immunotherapy seems to prolong survival.     

Histologic evaluation of L1210 leukemia in treated and untreated mice

Summary BDF₁ mice bearing L1210 leukemia were treated with chemotherapy or in combination with neuraminidase-treated cells and BCG. Histological evaluation was done on these mice at various intervals after therapy in order to determine the rate and extent of metastatic involvement in various tissues and organs. Results were compared to tumor-bearing mice which were not treated. In all animals, tissues were classed as having minimal involvement, moderate involvement or maximal involvement based on a scale of 0 through 4. Results indicated that: (a) mice which were long term survivors did not completely reject their tumor for weeks after treatment with chemotherapy and immunotherapy; (b) complete tumor rejection did not indicate a restoration of normal tissue integrity; and (c) failure of chemotherapy-immunotherapy had no consistent pathology, but was probably due to tumor distribution rather then tumor burden per se.This study was supported, in part, by Contract No. NO1-CB-43864 and Grant No. CA 14460 from the National Cancer InstituteThe Abbreviations used are: BCNU; 1,3-bis-(2-chloroethyl)-1-nitros-ourea; Saline: 0.9% NaCl solution; BCG: Bacillus Calmette-Guerin; C. parvum: Corynebacterium parvum; pfu: plaque-forming units     

Post-operative intrapleural BCG in lung cancer: A 5-year follow-up report

Summary Of a total study group of 99 patients 56 received intrapleural BCG in three different dosages (5 patients 16×10⁶, 25 patients 32×10⁶, and 26 patients 64×10⁶ culturable particles) following resection treatment for lung cancer. This study group was compared with an historical control population, very closely matched with regard to age, sex, stage of disease, histology and type of operation.Although the clinical condition of the patients selected for BCG treatment was above average, no survival benefit ensued from the intrapleural BCG. On the contrary, disease-free survival in BCG receivers with stage I and II squamous cell carcinoma was shortened significantly at 2 years follow-up due to the earlier appearance of local recurrences, with the same tendency still present after 5 years. This observation suggests an enhancement of tumour growth by the intrapleural BCG treatment. This alarming phenomenon could be a warning to clinicians when planning clinical immunotherapy trials to be aware of the potentially deleterious effects of such treatment.     

A new side effect of BCG immunotherapy — BCG-induced arthritis in man

Summary Ten of 159 patients showed arthritic symptoms during the course of BCG immunotherapy for advanced cancer. The arthritic symptoms occurring after BCG injections had the following characteristics: (1) The incidence of arthritis was closely correlated with the host immunologic responsiveness to BCG; (2) These symptoms usually occurred 1–5 months after the first BCG injection (7/10); (3) The arthritic symptoms usually started with morning stiffness (9/10), which was followed by acute inflammatory signs in the affected joints. They gradually subsided in response to treatment with nonsteroid antiinflammatory drugs, but were not completely cured while the effectiveness of BCG continued; (4) The symptoms were aggravated by additional BCG injections (8/10). (5) This form of arthritis could be differentiated from rheumatoid arthritis, tuberculous, or purulent arthritis by its clinical course and by roentgenograms of the affected joints. It is thought to be induced by the adjuvant effect of BCG, and is a new side effect of BCG immunotherapy.     

Decrease of body temperature after aglepristone treatment in bitches

Body temperature responses and the timing of abortions were evaluated in pregnant bitches with the anti-progestin aglepristone. Fifteen purebred and crossbred, 25-45 days pregnant, were included in this study and seven untreated bitches at the same stage of pregnancy served as controls. Treated bitches were administered two applications of aglepristone (10 mg/kg SC) 24 h apart for pregnancy termination. Pregnancy termination was confirmed by ultrasonographic assessment. Body temperature was rectally measured three times a day for 6 days beginning 24 h before treatment or pregnancy diagnosis in the treated and control bitches, respectively. Additionally, serum progesterone concentrations were assessed at time points during the study in the treated bitches. Pregnancy was terminated in 14 treated bitches in a mean+/-S.E.M. of 4.3+/-0.7 days after treatment. Control bitches remained pregnant. In the treated bitches, but not in the controls, body temperature significantly decreased 24 h after the beginning of the treatments (P < 0.01) and then gradually returned to pre-treatment values. Correlation between the day of mean minimum body temperature and the day of pregnancy termination was low (0.07; > 0.05). Progesterone did not show significant change throughout the study. Body temperature does not seem to be a suitable variable to clinically monitor the aborting effect of aglepristone. Decrease of body temperature after aglepristone treatment could represent further evidence of its hypothalamic effects.     

Improvement of monocyte chemotaxis with BCG immunotherapy in acute lymphoblastic leukemia: Comparison of chemotherapy versus chemotherapy plus immunotherapy as therapeutic regimens

Summary Thirty-six patients with acute lymphoblastic leukemia (ALL) were divided into a group receiving chemotherapy and a group receiving identical chemotherapy plus BCG immunotherapy. Monocyte cellular chemotaxis in both groups was initially significantly depressed compared with that in a group of age-matched controls (P<0.001). Chemotaxis in both patient groups improved with therapy. The mean improvement in chemotaxis per day of the group treated with chemotherapy plus BCG was over 3 times that of the group receiving chemotherapy alone (P<0.01). This change was not due solely to quantitative increases in monocyte numbers. The T-cell subset of ALL patients showed the same response in chemotaxis to chemotherapy plus immunotherapy as the null-cell ALL patients. In vitro monocyte chemotaxis in ALL patients was significantly increased by the addition of BCG immunotherapy to a standard chemotherapy regimen.     

The effects of metergoline combined with PGF2alpha treatment on luteal function and gestation in pregnant bitches

This study was designed to determine the nature of the antiluteotrophic effect of metergoline on pregnant bitches, the occurrence of clinically evident side-effects, and the efficacy of PGF(2alpha) in initiating abortion after a course of metergoline therapy. Starting on Days 18 to 20 after the onset of diestrus, 8 adult pregnant beagle bitches were treated twice daily with 0.4 to 0.5 mg/kg po metergoline for 5 d. After receiving no treatment for the next 5 d, metergoline administration was repeated for a further 3 d, followed by twice-daily intramuscular injections of dinoprost tromethamine at 250 mug/kg. There was an overall trend for the plasma progesterone concentration to decrease during the first and second course of metergoline therapy and to rise during the intervening period of no treatment. None of the bitches aborted during or after the first 5 d of metergoline administration. No side-effects were evident during the metergoline therapy. After pretreatment with metergoline a mean of 4.8 injections of PGF(2alpha) was necessary to ensure complete abortion. All abortions were rapid and uneventful, except for the usual side-effects associated with PGF(2alpha). The plasma progesterone concentration at the onset of PGF(2alpha) treatment was positively correlated to the number of PGF(2alpha) injections needed to complete the abortion process. The plasma progesterone concentration was < 8 nmol/l for several days as a result of metergoline therapy in 6 of the 8 bitches. Only 1 bitch, however, aborted before PGF(2alpha) therapy was initiated. In the other 7 bitches PGF(2alpha) appeared to be necessary for abortion. The results suggest that the effect of metergoline has to be considered incompletely luteolytic at the doses used in this study. Even prolonged suppression of luteal function in early to mid-gestation, however, did not cause abortion without the previously documented luteolytic and/or ecbolic effects of PGF(2alpha). The average interestrus interval of cycles treated with metergoline and PGF(2alpha) was shorter (mean 182.2 d, SD 7.9 d, n = 6) than expected for this group of bitches (mean 211.4 d, SD 31.5 d, n = 7).     

Immunotherapy in a spontaneously developed murine mammary carcinoma with syngeneic monoclonal antibody

Summary A new spontaneously arisen murine breast tumor, designated JC, has been established in immunocompetent BALB/c mice. Upon reestablishment of tumor in vitro and in vivo, the epithelial murine tumor cells retained their original papillary adenocarcinoma morphology. Various immunotherapy protocols have been performed in previously implanted and progressively growing JC tumor in syngeneic hosts with a murine monoclonal antibody (McAb), F36/22 (IgG3). Affinity of McAb binding to JC tumor cells was determined to be 6.1×10⁷ L/M. Quantitatively 1.2×10⁵ molecules of McAb bound to a JC tumor cell. Immunotherapeutic effectiveness in vivo on a tumor mass after its establishment is a major feature of this experimental tumor model. When four sequential administration of McAb, i.p., at a dose of 400 g 4 days apart were used, McAb-treated animals showed statistically significant tumor regression and longer survival than those of control animals treated with an irrelevant McAb of the same isotype. Two temporal phases of tumoricidal activity were observed as measured by tumor volume reduction. The first phase of tumoricidal response (tumor regression) was detected within days upon the first administration of McAb. A distinct second phase followed within 3–5 weeks after the last McAb administration, which resulted in tumor necrosis even in large tumors. Histological examinations revealed heavy infiltration of inflammatory cells at the beginning of the second phase. Similar tumor regression was also obtained from animals treated with a single dose (400 g) of McAb followed by injections of McAb with complete and incomplete adjuvant, respectively. These results demonstrate that this syngeneic murine mammary tumor can serve as a potential preclinical model for investigation of parameters and mechanisms associated with McAb immunotherapy.     

Adjuvant BCG immunotherapy for stage I and II malignant melanoma.

Initial adjuvant immunotherapy trials have demonstrated a greater disease-free interval in patients treated with bacille Calmette-Guérin (BCG) compared with historical controls. In this study 149 patients at high risk of recurrence after surgical treatment of local or regional malignant melanoma were given BCG for 2 years and were followed up for a median of 28 months from the start of immunotherapy. The 36 patients in the comparison group had a higher rate of recurrence than the patients treated with BCG, and the rate in the treatment group was close to that reported from a similar study at the University of California at Los Angeles. The relatively long disease-free interval for the high-risk comparison patients in this study suggests that the control groups at other centres may have included patients with unrecognized additional risk. The rates of survival in the Canadian treatment group were also comparable to those reported by other centres. However, reports of a favourable BCG-mediated pattern of recurrence could not be confirmed. Therefore, the routine use of adjuvant BCG immunotherapy is not recommended.     

Immunotherapy with tumor cell subpopulations

Summary The use of isolated tumor cell subpopulations combined with nonspecific immunostimulation (BCG and C parvum) was studied in the L1210-B6D2F₁ tumor-host model. Some tumor cell vaccines abrogated the immunotherapeutic value of nonspecific immunostimulants. A tumor cell vaccine prepared from a subpopulation with no apparent immunotherapeutic value completely neutralized the excellent therapeutic value of C parvum in this tumor-host model. The results emphasize the limitations of immunotherapy, and also the need to understand fundamental relationships between the immunological status of the host and subsequent immune stimulation.     

Therapeutic application of various immunostimulators on the L2C guinea-pig leukemia

Summary Immunostimulators such as Corynebacterium parvum (C. parvum), Bacillus Calmette-Guerin (BCG), pyran copolymer, and glucan were examined in the guinea pig L ₂ C lymphoblastic leukemia model to determine their capacity for therapeutic modulation of the immune response of the host toward controlling leukemic cell proliferation. The dose, route, and frequency of administration of the stimulators were also evaluated as a function of time in order to obtain an optimal antileukemic effect. Results indicated that only C. parvum and BCG were capable of significantly increasing host survival when given 1 day after an inoculation of 1.5×10 ⁴ viable leukemic cells. Administration of BCG or C. parvum, alone or in combination with irradiated blast cells on either days 4 or 7, was totally ineffective in prolonging survival. In the majority of cases, enhanced leukemic growth was observed on these days. The combination of BCG and/or C. parvum with irradiated syngeneic blast cells given 24 h after leukemia inoculation promoted a synergistic response with a significant increase in median survival time and a number of long-term survivors.This work was supported by contract N01-CP-53566 within the Virus Cancer Program of the National Cancer Institute