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.

Susceptibility to carcinogenesis.

This report examines qualitative and quantitative evidence that the spontaneous incidence of a specific type of tumor is one of the many factors which determine the incidence of carcinogen-induced tumors of the same type. To avoid selection bias in the choice of data for analysis, all suitable data used in a previous review on a closely related subject are analyzed. One method of attack consists of the mathematical elimination of factors other than spontaneous incidence which influence carcinogenesis. When unselected carcinogenesis data are examined without such elimination, then the spontaneous incidence often has no apparent effect on induced incidence. The explanation appears to be that the spontaneous incidence is a weak factor which has incomplete penetrance, and frequently is outweighed by other factors. Although the evidence examined is consistent with this conclusion, this does not constitute proof. Regarding mechanism, a carcinogen is seen to enhance the potential for the conversion of normal progenitor cells into cancer cells, which underlies the spontaneous incidence of tumors. It follows that carcinogen assays should be most sensitive, when the type of tumor induced by the carcinogen has a spontaneous incidence which is overt but low, or is just barely subthreshold.

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.

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.

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.

Determination of carcinogenic synergism from the latent period.

We present a method for determining the presence or absence of synergism between two carcinogens A and B from the latent period of tumors. The method is applied to data consisting of the means and standard deviations of the times at which tumors become palpable and at which death from tumor occurs in animals of three groups: those given carcinogen A alone, those given B alone, and those given both A and B. Synergism was absent in the data examined.

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).

Susceptibility to cancer and spontaneous incidence.

Susceptibility to cancer implies being easily affected by carcinogen, as well as having an overt spontaneous incidence of cancer. The susceptibility of a population to the development of fatal cancer of a given organ can be represented by a frequency distribution. This distribution depends both upon the genetic susceptibility of the population and upon all environmental carcinogens that have impinged on that population. The method for construction of such a susceptibility distribution has been simplified and applied to experimental data on bone tumor induction with 90Sr in mice, and to bone tumor mortality and prostate cancer mortality in man. The relative susceptibilities of different human organs to the development of fatal tumors can be defined in terms of the spontaneous tumor mortalities.

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).

An explanation for the proportion of carcinomas and sarcomas seen in chemically induced murine submaxillary gland tumors.

Pellets of 1 mg 9,10-dimethyl-1,3-benzanthracene (DMBA) were implanted into the submaxillary glands of 53 male C57BL/6J mice, and groups of mice were autopsied weekly or biweekly thereafter. Histologic evidence of tumor was noticed first at 12 weeks. From 16 weeks onwards, the submaxillary glands of all mice autopsied contained either carcinoma (three animals) or sarcoma (ten animals); 8/9 attempts to transplant these tumors in C57BL/6J mice were successful. Of the resulting eight tumor lines, two carcinomas and two fibrosarcomas were transplanted for over 30 months. Within the first few transplant generations, all four tumors showed an increase in growth rate and in histologic evidence of anaplasticity. For the particular tumors selected for study, the two carcinomas differed from the two sarcomas by growing more slowly, requiring more cells for tumor takes, and possessing a higher immunogenicity. These results may explain the types of tumors generated in submaxillary gland carcinogenesis. Carcinomas appear first, since they develop from the epithelial cells into which the carcinogen is implanted. Later, when fibroblasts wall off the carcinogen, fibroblasts are at risk for neoplastic conversion. Because of the more aggressive nature of the resultant fibrosarcoma cells, sarcomas may overgrow some early carcinomas.

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.

Effect of cigarette smoking on susceptibility to lung cancer.

Fisher first suggested that genetic predisposition to lung cancer is a more important factor than cigarette smoking, and that therefore one may smoke. Recently, this thesis has been defended strongly by Burch. Here, a literature survey has been done that indicates that the genetic susceptibility for development of lung cancer varies between individuals. But it is wrong to conclude from this that therefore one may smoke. Such a conclusion only would be valid, if there was a close relationship between the genetic tendency to smoke cigarettes, and the genetic tendency to develop lung cancer. The evidence that such a relationship does not exist is overwhelming. It is, therefore, valid to conclude that the large excess of deaths from lung cancer in cigarette smokers as compared to nonsmokers is a direct consequence of smoking cigarettes. To illustrate the effect of cigarette smoking on susceptibility to lung cancer, the distribution of susceptibilities to lung cancer in cigarette smokers and in nonsmokers has been derived. The shape of the susceptibility distribution is determined by the effects of all environmental carcinogens (both known and unknown) to which the population has been exposed, as well as by differences in genetic susceptibility between members of the population. The method described has general application.

Skin sensitization with the new reagents NOPYE (4-nitro-1-cyclohexyl-3-ethoxy-2-oxo-3-pyrroline) and NOPY-L-phenylalanine.

The aim of this study was to investigate the compound 4-nitro-1-cyclohexyl-3-ethoxy-2-oxo-3-pyrroline (NOPYE) and some related compounds for skin sensitization in guinea pigs, as the first step in a search for more effective skin sensitizers for immunotherapy of cutaneous tumors. In guinea pigs, NOPYE and NOPYE-L-alanine produce far milder delayed hypersensitivity reactions than DNCB. Both NOPYE and DNCB fail to act as adjuvants for skin sensitization to tuberculin purified protein derivative (PPD) and ovalbumin (OV). This suggests an explanation for the lack of effectiveness of DNCB in immunotherapy of metastases: DNCB may be relatively ineffective as an adjuvant for production of specific antitumor immunity. Such adjuvant activity may be essential if the action of the immunotherapeutic reagent is not to be confined to its site of application but is to be effective at the site of distant metastases.