Serial immunologic assessment during a randomized trial of chemoimmunotherapy in acute myelogenous leukemia

Summary Thirty-one adults who had acute myelogenous leukemia and in whom remission had been induced and consolidated with chemotherapy were randomized to receive one of three maintenance schedules: (A) BCG + chemotherapy [1, 3-bis-(2-chlorethyl)-1-nitrosourea (BCNU) and cytosine arabinoside]; (B) splenectomy, followed 1 week later with BCG and chemotherapy; or (C) allogeneic leukemic cells, BCG, and chemotherapy. Serial immunologic assessments were performed at the onset of maintenance and every 3 months.No differences were found in duration of remission (median 209 days) or survival (median 454 days) among the three schedules. Six patients remain in remission after from 2–4 + years. Skin test responses, mitogen responses, mixed lymphocyte culture responses, antibody responses, and T and B lymphocyte numbers were depressed at the onset of maintenance therapy. Therapy clearly improved the state of anergy as defined by recall antigen responsiveness, and induced in vivo and in vitro PPD reactivity. However, immunotherapy resulted in a reduction of the number of T or B cells and of the in vitro lymphocyte response to mitogens and allogeneic cells. Serum obtained at diagnosis and during remission inhibited in vitro blastogenic responses in more than half the patients. These data indicate that chemoimmunotherapy given as described tended to be more immunosuppressive than stimulatory.     

Immunologic function during adjuvant BCG immunotherapy for malignant melanoma: Induction of anergy

Summary Serial tests of immunological function were performed on 28 patients participating in a randomized controlled clinical trial of adjuvant Tice-stain BCG immunotherapy administered by tine technique for malignant melanoma. Cryopreserved lymphocyte samples obtained prior to study entry and at 3 and 6 months there-after were tested by mixed lymphocyte culture (MLC), cell-mediated lympholysis (CML), antibody-dependent cell-mediated cytotoxicity (K cell), and natural killing (NK cell) assays, the last two assays being performed with the Chang cell line. Delayed-type hypersensitivity (DTH) skin tests to recall antigens were performed at the same intervals.At entry to the study in vitro lymphocyte reactivity of patients was similar to that of normal controls, and most (75%) of the patients reacted to at least one recall antigen. Serial lymphocyte reactivity measured by the in vitro tests was not different in the BCG and control groups, but BCG treatment was associated with a marked, statistically significant (P<0.01) reduction in DTH skin test reactivity. BCG therapy was not shown to delay recurrence of the disease.     

Active specific immunotherapy of Dukes B2 and C colorectal carcinoma: Comparison of two doses of the vaccine

Summary The ability of active specific immunotherapy to enhance immune responses to autologous tumor-associated antigens (TAA) and to prolong the disease-free interval was evaluated in patients with Dukes B₂ and C colorectal carcinoma who had undergone potentially curative resections. Patients were sensitized in the early postoperative period with irradiated autologous adenocarcinoma cells mixed with bacillus Calmette-Guérin (BCG) to yield either a low-dose vaccine (3×10⁶ tumor cells) or a high-dose vaccine (1×10⁷ tumor cells). Six of seven patients who received the low-dose vaccine developed delayed-type hypersensitivity (DTH) responses to autologous tumor cells upon completion of the vaccination, whereas all four patients receiving high-dose vaccine displayed a positive DTH response. However, DTH responses to autologous TAA waned within 3 months in all patients receiving the low-dose vaccine; DTH responses persisted for 3 months in three of the four high-dose vaccine patients. In vitro lymphoproliferative responses to TAA correlated with DTH responses to autologous tumor cells. Active specific immunotherapy appeared to induce specific immune responses either in vitro or in vivo to autologous TAA because it did not induce responses to autologous mucosa cells. There were no complications caused by BCG or tumor cells. This series demonstrates that active specific immunotherapy is a nontoxic treatment that augments immunity to autologous TAA.This project was supported by grants from Cutter Laboratories, Inc., the Annual Campaign of the University of Texas System Cancer Center, and the National Cancer Cytology Center     

Immunologic approaches to the treatment of human cancer based on a guinea pig model

Summary Local immunotherapy is a form of cancer treatment where exogenous antigen is introduced into the area of the tumor. Under favorable circumstances, the perfused tumor regresses, systemic tumor-specific transplantation immunity is augmented, and distant microscopic metastases regress. Successful local immunotherapy requires an immunologically competent host, small tumor burden, and tumor located usually in the skin. A wide variety of biologic agents are capable of promoting local immunotherapy. BCG has been most widely studied. The antitumor activity of two different preparations of the Tice substrain of BCG were compared. No significant differences in antitumor activity were found. Alternative approaches to intralesional injection were sought. Intradermal injection of BCG adjacent to dermal tumors, prior to surgery, led to eradication of axillary metastases and to the development of tumor-specific transplantation immunity.Successful local BCG immunotherapy is a by-product of the host response to BCG infection. Involved are lymphocytes specifically sensitized to mycobacterial antigens, lymphocyte mediators, and macrophages which develop the capacity to kill tumor cells. Tumor-cell killing may be mediated by exocytosis of macrophage lysosomes into tumor cells. Complete and permanent tumor eradication probably requires the development of tumor-specific transplantation immunity mediated by sensitized lymphocytes. Local infection of the tumor may augment the development of this tumor-specific immunity.     

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     

Effects of immunotherapy and chemotherapy on immunocompetence. A study of patients with acute myeloblastic leukemia in remission

Summary The immunocompetence of 33 patients with acute myeloblastic leukemia in remission and treated with cytostatics (CT) was studied. In addition to cytostatics some of the patients were given immunotherapy (CT+IT).In an attempt to demonstrate immunization against allogeneic leukemic blast cells (or their extracts) or immunostimulation after immunotherapy or, alternatively, immunodepression after maintenance chemotherapy without immunotherapy, delayed hypersensitivity tests and lymphocyte stimulation tests were performed. In most cases PHA seemed to be a stronger stimulator than allogeneic lymphocytes and these seemed to be stronger than allogeneic blasts, although no difference was statisically significant.No significant differences were found in vitro or in vivo between the reactions of CT and CT+IT patients or their lymphocytes to allogeneic myeloblasts or to allogeneic lymphocytes. However, numerically, in vitro and in vivo CT+IT patients reacted more to myeloblasts, CT patients more to lymphocytes. This could suggest antigens on leukemic myeloblasts that are not found on lymphocytes. With present methods we could demonstrate neither immunodepression in patients given only chemotherapy nor nonspecific immunostimulation after immunotherapy. There was no significant difference between the two treatment groups in lymphocyte reactivity against PHA and allogeneic lymphocytes. Nor was the lymphocyte reactivity different from that in a group of healthy persons.Decreasing lymphocyte reactivity to PHA and allogeneic lymphocytes seemed to herald relapse.     

Conversion of human lymphocytes to antitumor immune reactivity by xenogeneic and allogeneic imune RNA detected by leukocyte-adherence inhibition tests

Summary Using leukocyte adherence inhibition (LAI) tests, we studied the activity of xenogeneic immune RNA (I-RNA) extracted from the spleen and lymph nodes of sheep after immunization with human breast carcinoma tissue or keyhole limpet hemocyanin (KLH) in inducing lymphocytes from normal healthy donors to mediate immune responses in vitro. Mononuclear cells isolated from venous blood of normal donors, depleted of monocytes and, in some experiments, separated into T cells and non-T cells, were incubated with and without anti-breast carcinoma I-RNA or anti-KLH I-RNA for 20 min at 37° C. Then, lymphocyte adherence was determined by a Coulter counter method in the presence of 3 M KCl extracts of breast carcinoma tissues, control tissue, or KLH. Following incubation with anti-breast carcinoma I-RNA, the adherence of lymphocytes from normal donors was found to be inhibited only in the presence of breast carcinoma extracts. Following incubation with anti-KLH I-RNA, lymphocyte adherence was inhibited only in the presence of KLH. The principal effector cells involved appeared to be T lymphocytes. I-RNA treatment with RNase, but not with DNase or pronase, completely abrogated the LAI responses. In a blind study utilizing coded samples of xenogeneic and allogeneic I-RNA of unknown origin, samples containing activity against breast cancer extracts were identified correctly by LAI. Abbreviations used: I-RNA, immune RNA; LAI, leukocyte adherence inhibition; KLH, Keyhole limpet hemocyanin; PBS, phosphate buffered saline; RNase, ribonuclease; DNase, deoxyribonuclease     

BCG immunotherapy in stage I melanoma patients

Summary Previously, we have provided evidence for a positive correlation between HLA-DR expression in primary melanoma and early metastasis [3, 4]. In the present study we investigated whether this relationship was modified by adjuvant BCG immunotherapy. The study comprised 107 patients with a stage I high-risk melanoma; 44 patients had been treated with BCG, whereas the remaining patients had not received any adjuvant therapy. There was no difference in disease-free survival between BCG-treated and untreated patients. Disease-free survival was significantly shorter in patients with high expression of HLA-DR antigens in the primary tumor.Subgrouping BCG-treated and control patients according to HLA-DR phenotype of the melanoma revealed a prolongation of disease-free survival in the subgroup of BCG-treated patients with no or low expression of HLA-DR antigens in the primary melanoma. BCG therapy apparently did not influence prognosis of patients with high expression of HLA-DR antigens in the tumor.     

Immune reactivity in cattle with ocular squamous cell carcinoma after intralesional BCG immunotherapy

Summary Lymphocyte stimulation with Con A and specific immune reactivity to BCG (antibody formation to BCG and DTH reaction to PPD) were determined in BCG-treated, surgically treated and untreated cows with ocular squamous cell carcinoma. In tumor-bearing cows the Con A-induced proliferation of lymphocytes was reduced when compared to healthy controls. This suppression consisted of a reduced blastogenic response to Con A of lymphocytes from tumor-bearing cows, and the presence of a factor in the sera of these animals, as these sera suppressed the blastogenic response of lymphocytes from healthy cows. BCG had only a minor influence on the suppressive activity. Antibodies to BCG were demonstrated in 50% of the BCG-treated animals. The formation of antibodies was not influenced by intradermal injection of PPD of Mycobacterium bovis. Absorption of a BCG antibody containing serum with BOSCC tumor extracts did not reveal the existence of cross reacting antigens between BCG and BOSCC. Pretherapeutic and posttherapeutic Con A reactivity could not be correlated with clinical response. Of the 30 BCG treated cows 29 developed a positive DTH reaction to PPD. Correlation between clinical response and immune reactivity was seen only with regard to the DTH reaction to PPD: this reaction remained positive for a longer period after treatment in animals with a favorable clinical outcome than in nonresponding animals.Animals were maintained under the guidelines laid down by the Faculty of Veterinary Medicine, State University, Utrecht, The NetherlandsGrant recipient of the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation) Abbreviations used: BCG, Bacillus Calmette-Guerin; BOSCC, bovine ocular squamous cell carcinoma PBL peripheral blood leukocytes; PPD, purified protein derivative of Mycobacteria; DTH, delayed type hypersensitivity Con A, concanavalin A; PHA, phytohemagglutinin; PWM, pokeweed mitogen     

Shared antigens between human malignant melanoma cells and Mycobacterium bovis (BCG).

This study was undertaken to investigate the antigenic relationships between human malignant melanoma cells and Mycobacterium bovis (BCG). Rabbits were immunized with sonicates of BCG or with malignant melanoma cells from different patients and the resulting antisera were tested for their capacity to bind radiolabeled soluble extracts prepared from BCG and melanoma cells. The binding of antibodies to radiolabeled antigens was studied by precipitation of radiolabeled antigen-antibody complexes by anti-rabbit immunoglobulin. Antibodies in sera from rabbits immunized with either BCG (anti-BCG) or melanoma cells (anti-melanoma) bound both the labeled BCG and melanoma antigens. Control antisera, from rabbits immunized with human acute or chronic lymphatic leukemia cells or with normal human spleen cells, did not bind significant amounts of radiolabeled BCG. Antibodies in sera from rabbits immunized with normal spleen cells bound small but significant amounts of radiolabeled melanoma antigens. Binding by anti-BCG and anti-melanoma to the radiolabeled antigens was studied before and after absorption of antisera with cells from human melanoma, leukemia, guinea pig hepatoma, and normal human spleen cells. Inhibition studies using unlabeled BCG extracts also were carried out. The absorption and inhibition studies confirmed that the binding reactions were specific and that antigens from five melanoma patients shared antigenic determinants with BCG.     

Experimental screening of BCG preparations produced for cancer immunotherapy: Safety and immunostimulating and antitumor activity of four consecutively produced batches

Summary Four consecutively produced batches of Bacillus Calmette-Guérin (BCG) especially intended to be used for cancer immunotherapy were investigated for consistency of the vaccine. Each batch was investigated directly after production of the vaccine, so that the four batches were not tested simultaneously. The activity of the four batches was investigated in general safety assays, immunostimulation assays, and two different tumor models.General safety assays showed dose-dependent growth retardation and increased serum glutamic pyruvic transaminase activity in mice, and a long-lasting temperature rise in rabbits after IV administration of the BCG preparations. In a skin reactivity assay, reactions were found acceptable for all preparations when compared with a reference batch.The results of the immunostimulation and antitumor studies can be summarized as follows. All four batches induced a specific delayed-type hypersensitivity reaction to PPD, indicating the induction of cell-mediated immunity. A stimulating effect on lymphoreticular organs was concluded from increased spleen weight and enhanced cell proliferation in draining lymph nodes. Enhanced macrophage function (phagocytosis and killing of bacteria) was demonstrated by an increased resistance to Listeria monocytogenes. YAC lymphoma target cells were killed nonspecifically by BCG-activated peritoneal exudate cells (PEC), indicating the induction of natural killing activity by BCG.Intralesional injection of BCG induced tumor regression in the guinea pig line 10 hepatocellular carcinoma, followed by immunity to the line 10 tumor. In the murine 5D04 squamous cell carcinoma, BCG had no effect on the primary tumor. However, IV-injected BCG resulted in a decreased number of lung metastases.In general, the four consecutively produced batches showed similar safety and activity in the immunostimulation assays and antitumor activity. Since only minor differences between the batches were found, which can also be attributed to the variation in experimental conditions common to biological assays, it is concluded that the vaccine batches produced show an acceptable consistency.Abbreviations used BCG, Bacillus Calmette-Guérin; C. parvum, Corynebacterium parvum; c. p., culturable particles; IU, international unit; PPD, purified protein derivative; PEC, peritoneal exudate cells     

Assessment of cell-mediated immunity in the rat utilizing the agarose-droplet cell-migration-inhibition correlate of delayed-type hypersensitivity.

The agarose-droplet cell-migration-inhibition assay was developed for measuring specific cell-mediated immunity in rats. Fischer #344 female rats were sensitized to mono(p-azobenzene-arsonate)-N-chloroacetyl-L-tyrosine (ARSNAT), keyhole-limpet-hemocyanin (KLH), or a BCG cell wall preparation. The optimal conditions for assay were determined by testing varying concentrations of antigens against normal and sensitized peritoneal exudate cells induced with 5% thioglycolate medium. Specific cell-mediated immunity to each of three different antigens was detected, which correlated with skin tests observed $\text{in vivo}$. Our adaptation of the agarose-droplet assay should provide a useful model for studying other aspects of cell-mediated and/or tumor immunity in the rat.     

Direct endolymphatic instillation (ELI) of BCG-Tumor cell mixture

Summary A method of active immunization by direct Endolymphatic Instillation (ELI) of a mixture of BCG and Autologous Irradiated Tumor Cells (AITC) is described. ELI was performed in 36 patients with advanced breast cancer, melanoma and some other solid tumors. The procedure was well tolerated and was not associated with any untoward systemic side effects other than those often encountered in conventional BCG immunotherapy. Exposure of patients to AITC by the direct endolymphatic route proved to be superior to immunization by the intradermal route as judged by the subsequent induction of cutaneous Delayed Hypersensitivity Reaction (DHR) toward AITC. When employed as the first immunization attempt ELI was effective in 5 of 6 patients. It led to invigoration of preexisting weak DHR in 6 of 8 patients. In 15 of 22 patients conversion to a strongly positive cutaneous DHR to AITC was achieved by ELI, where previous attempts of immunization by the intradermal route had failed to elicit a positive response. Acquisition of positive cutaneous response to AITC following ELI was found to correlate with favourable prognosis in terms of longer median survival. The effect of ELI was particularly notable with respect to enhancement of in vitro lymphocyte stimulation by PPD antigen, but was not followed by increased reactivity of other in vitro parameters of cell-mediated immunity such as peripheral lymphocyte count, proportion of E-rosette forming cells, and PHA lymphocyte stimulation.Abbreviations used: ELI — Endolymphatic Instillation; AITC — Autologous Irradiated Tumor Cells; BCG — Bacillus Calmette-Guérin; DHR — Delayed Hypersensitivity Reaction; PPD — Purified Protein Derivative (tuberculin); PHA — Phytohemagglutinin; SI — Stimulation Index; E-RFC — E-Rosette Forming Cells; PBL — Peripheral Blood Lymphocytes.     

Echinococcus granulosus: host lymphocyte transformation by parasite antigens.

Lymphocyte transformation, measured by in vitro tritiated thymidine incorporation, and indirect hemagglutination tests were carried out on hydatid patients and normal individuals using sheep and human hydatid fluid or scolex antigens. The hydatid patients showed statistically significant lymphocyte transformation with human and sheep hydatid fluid or scolex antigens when compared to normal individuals. The indirect hemagglutination tests resulted in high titers of antibody with sheep or human hydatid fluid antigens, while very low titers were obtained with scolex antigens. Unlike in the indirect hemagglutination test, the source of the antigen, scolex or fluid, was not of consequence in the lymphocyte transformation test. Furthermore, there was no correlation between the results of the serologic and lymphocyte transformation tests, since some patients with very high lymphocyte stimulation indices produced low indirect hemagglutination titers and vice versa. Similar results were obtained from rabbits which were immunized with sheep hydatid fluid or scolex extracts. The skin tests were of the immediate type of hypersensitivity reactions. Delayed skin reactions did not occur in spite of the presence of sensitized lymphocytes in the blood of the immunized rabbits.     

The immunopathological effects of intracolonic injection of Mycobacterium bovis BCG cell wall emulsions in guinea pigs

Summary The immunological and pathological responses of guinea pigs to an intramural colonic injection of emulsions containing cell wall (CW) extracts of Mycobacterium bovis BCG and mineral oil were studied from week 1 to week 36 post-inoculation. The emulsions contained variable concentrations of BCG CW attached to (lipid-phase), or separate from (aqueous-phase), the mineral oil. Delayed cutaneous hypersensitivity to PPD was present throughout the course of the study in a variable percentage of guinea pigs inoculated with either type of emulsion. PPD-induced blast transformation of peripheral blood lymphocytes, studied in guinea pigs which received lipid-phase emulsion, was also detectable throughout the course of the study, with maximal response seen 2 weeks post-inoculation. The intracolonic inoculations were well tolerated, with the exception of the most concentrated lipid-phase emulsion (3 mg/ml BCG CW and 5% oil), after which one of eight guinea pigs died due to a colonic impaction and rupture at the site of inoculation. The pathological response to either type of emulsion was a focal granulomatous colitis, which tended to be more severe as the concentration of BCG CW and oil increased. Extracolonic lesions were usually limited to a granulomatous lymphadenitis of lymph nodes draining the injection site; however, the most concentrated lipid-phase emulsion occasionally produced granulomatous inflammatory foci in the liver and lungs. In general, the lesions induced by the lipid-phase emulsions were more severe than those induced by aqueous-phase emulsions, but the intensity of both types of lesions peaked at 2 or 4 weeks post-inoculation. It was concluded that the guinea pig may serve as a useful model to study BCG immunotherapy of colonic neoplasms, since intracolonic injection of BCG CW resulted in systemic immunity toward mycobacterial antigens and a localized accumulation of macrophages without untoward complications. The abbreviations used in this paper are: BCG CW, bacillus Calmette-Guérin cell walls; PPD, purified protein derivative; PBL, peripheral blood lymphocytes; cpm, counts per minute; SI, stimulation index; DCH, delayed cutaneous hypersensitivity     

Cancer immunotherapy.

Important contributions that stimulated studies in cancer immunotherapy included: (1) the discovery of tumour-associated antigens; (2) the observation that infection with bacille Calmette-Guérin (BCG) in animals was protective against tumour challenge; and (3) the observation that immunodepression due either to malignant disease or to treatment of the disease, was, in some instances, related to prognosis. Immunotherapy trials with microbial agents have involved attempts to obtain a local effect by injecting the agent into the tumour or into the region of the tumour and to obtain a "systemic" effect distant from the site of injection. Trials with active specific immunotherapy involving tumour cells or tumour cell extracts have frequently involved the combination of these specific agents with a nonspecific adjuvant such as BCG. Recent studies with thymosin and levamisole in patients with lung cancer and other types of malignant disease have shown prolonged survival in the groups receiving immunotherapy.     

Immunologic responses in patients with pulmonary carcinoma receiving immunotherapy

Summary This is a preliminary report on immune responses observed in vivo and in vitro in resectable patients treated with oral BCG, and in nonresectable patients treated with autologous tumour cells complexed with PPD and with BCG applied by scarification. In some cases, to shorten the period of sensitization, transfer factor was given additionally. Several of the in vitro methods used for assessing the immune competence of the patients are shown to be sufficiently informative to warrant their use before immunotherapy in order to assess the immunologic resources of the patient. During immunotherapy, they allow monitoring the effect of the treatment.     

Rapid effects of BCG on T cell function in cancer patients

Summary Forty-four cancer patients were evaluated with multiple sequential tests to determine the effects of a single intracutaneous injection of BCG on the levels of total and high-affinity sheep erythrocyte-forming cells and on lymphoproliferative responses to a T cell mitogen (PHA) and antigens (MLC and PPD). The analysis of the data was complicated by considerable pretreatment variation in the results with some patients and by the lack of a consistent change in responses among all patients. However, exploratory statistical procedures made it possible to demonstrate that inoculation with BCG was associated with a significant transient increase, on day 3 post-injection, in levels of total and active rosette-forming cells and in lymphoproliferative responses to PPD. The effects on responses to PHA varied with the level of pretreatment reactivity with patients with initial low responses showing a significant increase on days 3 and 7 and patients with initial normal responses showing a significant decrease on days 3 and 7. Patients and normal individuals receiving no injection, and patients receiving isotonic saline injections, showed none of these changes. Thus it appears that BCG may cause measurable changes in T cell levels and lymphoproliferation to T cell mitogen (PHA) and antigen (PPD).     

Active,specific immunotherapy of stage III breast cancer

Summary Active specific immunization with autologous irradiated tumor cells (AITC) admixed with BCG was attempted in 49 stage III breast cancer patients whose median follow-up at present is 3 years. As a first immunizing procedure 41 patients received repeated intradermal inoculations and 8 had a single endolymphatic instillation (ELI). Skin response (SR) to AITC alone was induced after two to nine weekly immunizations. Eight of 41 patients with negative or weak responses were effectively reimmunized by ELI, as indicated by conversion and invigoration of SR. Following immunization, radiotherapy to breast and axilla was administered. Thereafter, fortnightly 5-FU and monthly boosters of AITC-BCG mixture were given for 2 years. Strength of response to AITC induced by active immunization was found to relate to subsequent disease recurrence, with 15% relapsing among the good responders and 53% among the weak and non-responders. Positive SR to AITC — once elicited — was steadily maintained in the majority of patients; its decline was associated with manifest disease recurrence. Conversion to AITC positivity in vivo following specific immunization was not detectable by the LMI assay. Lymphocyte stimulation (MLTI) by AITC in vitro was found in only 9 of 26 tested patients with positive in vivo SR to AITC. Cutaneous response to AITC appears to be the only parameter of antitumor response showing clinical correlation, while specific in vitro correlates of cell-mediated immunity were found unsuitable for monitoring patients undergoing specific immunotherapy. While in vivo PPD response was mainly unchanged or enhanced, in vitro lymphocyte stimulation by PPD and PHA showed a distinct decline at the time of relapse. The cumulative proportion of relapse among the immunotherapy patients at 3 years was 32% with mortality of 12% (13 relapsed, 5 died), both being significantly lower than reported results in stage III breast cancer without immunotherapy. It is concluded that specific immunization with AITC is feasible in most breast cancer patients with loco-regionally advanced disease and that this intervention is conducive to favorable modification of the course of their disease.     

Cellular immunity in patients with multiple primary tumors

Summary Immunological studies for evaluation of cellular immunity were carried out on 22 patients who had two primary malignant tumors and who had no evidence of active disease. The tests performed were for skin reactivity to 3 recall antigens, PPD, Candidine, and Streptokinase and in vitro lymphocyte stimulation by PHA. Of these, 6 patients were anergic to all three recall antigens and 5 other patients to two out of three antigens. A depression of the in vitro reactivity was encountered in 11 patients. The immune incompetence was more evident in patients with simultaneous tumors than in those with metachroneous tumors. The longer the interval between the appearance of the primary and the secondary tumors, the less immunosuppression was seen.Presented at the 24th Israel Medical Association Conference on Immunological Aspects of Cancer, Tel-Aviv, December 26, 1974This work was supported in part by the Office of the Administrator General, Ministry of Justice and the Medical Research Fund under the Sponsorship of the Ministry of Health