Synergism in carcinogenesis.

Six examples of exposure of humans to two carcinogens were found that satisfied the minimum requirements for analysis of whether the carcinogens acted in synergism. In all instances, the risk ratio for cancer in the group exposed to both carcinogens was equal to or greater than the product of the risk ratios of the singly exposed groups. These findings underline the danger of double exposure, even though they imply that some and not all pairs of carcinogens act in this way. Because the six studies did not extend to the end of the life-spans of the exposed individuals, the data may be skewed by the life-shortening effect of promoters. Nevertheless, the findings have implications regarding the employment of people who already having been exposed to one recognized carcinogen are in a job where they may receive additional exposure from a second carcinogen known to multiply their risk of cancer. Analyses of animal tests on synergism indicated that most published studies lack dose-response data for each of the two carcinogens, which are vital for definitive proof of synergism. Suggestions for performance of such tests are given. The theoretical basis underlying the concept of synergism is examined, equations for its evaluation are given, and examples of statistical tests for its presence are presented. The conclusion regarding the mechanism of action was that synergism occurs if the rate-limiting step in the generation of a single type of tumor differs for each of two interacting carcinogens.

Relationship of success in classical immunotherapy to the relative immunorejective strength of the tumor.

Six tumors of varying immunorejective strengths were used to compare the response of their isogenic hosts to standardized regimens of immunoprophylaxis and immunotherapy. The tumors were generated spontaneously or induced chemically [with 9,10-dimethyl-1,2-benzanthracene (CAS: 57-97-6)], virally (with murine leukemia virus), or radiogenically (with strontium-90). The hosts were C57BL/6J or BALB/cByJ mice. Immunoprophylaxis and immunotherapy were performed with isogenic irradiated tumor cells, with Corynebacterium parvum, or with both. The results of challenge experiments were quantified as the doses of viable tumor cells that produced 50% tumor deaths for immunized and for control mice. The results for these quantitative "classical" immunoprophylaxis and immunotherapy experiments were consistent with two theses: that only immunorejective tumors give positive results with classical immunotherapy and that classical immunoprophylaxis is more effective than classical immunotherapy when identical materials are used for immunizations. These results have important consequences for the clinical use of classical immunotherapy.

Vaccination against strontium-90-induced bone tumors.

The thesis was tested that immunization against a murine osteosarcoma virus can reduce the incidence of bone tumors induced by 90Sr. C57BL/6J female mice (190) were divided into three sets of 2 groups. Each set consisted of a control group and an experimental group treated ip with 1.0 muCi 90Sr at 66 days of age. The three sets of groups received the following additional treatments: none (controls), 6 injections of Formalin-inactivated FBJ osteosarcoma virus (vaccinated group), or 6 injections of active FBJ virus (active virus controls). Only 1 bone tumor developed in a mouse not treated with 90Sr in the active virus controls. In 90Sr-treated mice, vaccination reduced bone tumor deaths during the first 600 days from 9 of 36 in controls to 1 of 33 in vaccinated mice (P less than .01), but bone tumor deaths during the entire life-span, 10 of 36 and 5 of 33, respectively, were not significantly different (P = .07). Thus the vaccination procedure delayed the development of bone tumors. In contrast, injection of active virus into 90Sr-treated mice increased the lifetime incidence of bone tumors from 10 of 36 in controls to 19 of 32 (P = .01).

Vaccination of adult and newborn mice of a resistant strain (C57BL/6J) against challenge with leukemias induced by Moloney murine leukemia virus.

Adult or newborn C57BL/6J mice were immunized with isogenic Moloney strain MuLV-induced leukemia cells irradiated with 10,000 rads or treated with low concentrations of formalin. Groups of immunized and control mice were challenged with a range of doses of viable leukemia cells, and tumor deaths were recorded for 90 days after challenge. Then, the doses of challenge cells which produced 50% tumor deaths were calculated for immunized and control mice. The logarithm of their ratio quantified the degree of protection provided by immunization. For adult C57BL/6J mice, a single immunization with MuLV-induced leukemia cells was not effective; either cells plus Bacillus Calmette-Guérin or Corynebacterium parvum, or else two immunizations with irradiated leukemia cells were needed to produce statistically significant increases in the values of the doses of challenge cells which produced 50% tumor deaths. Cross-protection was obtained by immunization with other isogenic MuLV-induced leukemias, but not by immunization with isogenic carcinogen-induced tumors or with an isogenic spontaneous leukemia. For newborn mice, a single injection of irradiated leukemia cells provided 1.3 to 1.5 logs of protection, and admixture of B. Calmette-Guérin or C. parvum increased this protection to 2.4 to 2.7 logs. Since irradiated and frozen-thawed MuLV-induced leukemia cells contained viable MuLV, leukemia cells treated with 0.5 or 1.0% formalin were tested as an alternative. A single injection of formalin-treated isogenic leukemia cells admixed with C. parvum provided between 1.7 and 2.8 logs of protection. These results demonstrate that a single vaccination of newborn animals against a highly antigenic virally induced leukemia produces strong protection against a subsequent challenge with viable leukemia cells.

Lack of suppressor cell activity in the spleens of mice with radiation-induced osteogenic sarcomas.

C57BL/6J mice were used both for induction of osteogenic sarcomas by injection of 90Sr and for induction of sarcomas and carcinomas by injection of 9,10-dimethyl-1,2-benzanthracene. The osteogenic sarcomas had a relatively long induction period; they possessed low immunogenicity and failed to activate splenic suppressor cells either in the original tumor-bearing host or in mice bearing transplanted osteogenic sarcomas. In contrast, and in agreement with previous work, 9,10-dimethyl-1,2-benzanthracene-induced tumors had a relatively short induction period; they possessed high immunogenicity and activated splenic suppressor cells in mice bearing transplanted tumors. These results suggest the possibility that the immunogenicity of tumors correlates with the ability of tumors to activate suppressor cells.

Relationship of macrophage content, immunogenicity, and metastatic potential of a murine osteosarcoma of recent origin.

The purpose of these studies was to determine whether the high macrophage content (greater than 50 per cent) of the 90Sr-induced osteogenic sarcoma J (Os-J) of recent origin correlated with its immunogenicity or low metastatic potential. Cloning experiments demonstrated that the Os-J tumor is heterogeneous with regard to the production of experimental pulmonary metastases. Immunization-challenge studies in syngeneic mice and comparisons of tumor growth in normal or nude mice established that the slow growing Os-J tumor is poorly immunogenic. In vitro studies demonstrated that the Os-J tumor is highly susceptible to macrophages-mediated lysis. This may explain the slow growth of the tumor in normal recipients with an intact mononuclear phagocyte system, as compared with the more rapid emergence of tumors in macrophage-suppressed mice. However, spontaneous metastases of the Os-J tumor were not observed either in normal or macrophage-suppressed mice. Although a high macrophage infiltration of neoplasms could slow tumor growth, this was not associated with the immunogenicity of the neoplasm and did not appear to limit the spontaneous metastasis of this essentially benign neoplasm.

'Sufficient' absorption--a quantitative method to replace 'exhaustive' absorption.

At present, the only guideline for removal of undesired reactivities from an antiserum is that no activity against the cross-reacting (undesired) antigens should remain after exhaustive absorption. Since this guideline sets no upper limit on the amount of undesired antigens required for an exhaustive absorption, waste of possibly precious material and of time in performing multiple sequential absorptions can result. To minimize the quantity of antigen and of experimental work required for an absorption, a quantitative approach is suggested: the antiserum is test-absorbed with various amounts of the undesired antigens, and antibody activity still present against these antigens is then tested. The results can be plotted as a curve by use of Reif's modification of the Von Krogh equation. Thus, the exact amount of undersired antigens sufficient to remove all detectable reactivity against these antigens can be determined. The procedure is termed 'sufficient' rather than 'exhaustive' absorption, to stress that only detectable amounts (rather than every last trace) of undesirable antibodies have been removed. The nomenclat re 'exhaustive absorption' gives no indication that any practical attempt to achieve it can hardly escape having the same limitations as 'sufficient absorption'; it is therefore suggested that the misleading non-quantitative nomenclature 'exhaustive absorption' be eliminated from immunological terminology. 'Sufficient absorption' has been applied to the absorption of undesired (blood group) antibodies from rabbit antisera to CEA.

Preparation and therapeutic potential of rabbit antisera with "directed" specificities for mouse leukemias.

Several immunization regimens for preparation of ALK-NABS were compared. One series of eight intravenous injections spaced over 5 weeks gave an ALK-NABS with potency and specificity that could be bettered only slightly by a second series of four injections spaced over 2 weeks, whereas a third series of injections was deleterious. Use of late immune antisplenocyte NABS for such immunizations produced ALK-NABS reagents with the highest in vitro specificity to leukemia cells relative to splenocytes after absorption, whereas early immune antisplenocyte NABS gave ALK-NABS with the highest antileukemia specificity relative to thymocytes. Therapy experiments with leukemias L1210 and BW-A showed increased survival times for isogeneic mice injected intraperitoneally with 10(3) (L1210 only), 10(4), and 10(5) (higher significance for L1210) cells, when ALK-NABS was given intraperitoneally in high dose on 4 or 5 successive days starting 1 day after inoculation of leukemia cells. In additional experiments with 10(5) cells given intraperitoneally, lower doses of ALK-NABS were progressively more effective with L1210 leukemia, producing some survivors without any apparent toxicity from the antiserum. In contrast, a similar experiment with leukemia BW-A was entirely negative. Addition of guinea pig serum to already excessive amounts of antiserum was not helpful.

Effects of strontium-90 plus external irradiation in C57BL/6J mice.

421 C57BL/6J female mice were subdivided into 11 groups. Five of these groups were given 300 rad total body irradiation from a 137Cs source at an age of 65 days. One day later, these irradiated mice were treated intraperitoneally with varying amounts of 90Sr (0, 0.032, 0.10, 0.32, and 1.0 mu Ci/g of body weight). Five groups of mice that had not been irradiated were treated on the same day with the same doses of 90Sr as given the five irradiated groups, and a sixth unirradiated group was treated with 2 mu Ci/g body weight. Each mouse treated with 90Sr and still alive was monitored between 249 and 303 days later in a total body well scintillation detector; mice with counts that differed by more than approximately 50% from the mean for their group were eliminated. A total of 402 mice were accepted for the experiment; these mice were followed to the end of their life span and then autopsied. Mice treated with the highest doses of 90Sr (1.0 and 2.0 mu Ci/g) experienced significantly elevated number of deaths from infections relative to the control group; these deaths occurred relatively early after 90Sr injection, and were particularly severe in the group of mice that had received 300 rad of external irradiation in addition to 1.0 mu Ci90Sr/g. There was no evidence of synergism between 90Sr injection and 300 rad external irradiation for production of bone tumors. Tumors of the type that occur spontaneously in C57BL/6J mice appeared to be more frequent in 90Sr-treated mice and in externally irradiated mice than in controls, but the numbers of excess tumors in these groups were not statistically significant (P less than 0.09).