This new classification is based on the following principles: Five basic histological patterns (adenocarcinoma, ca. epidermoides, ca. simplex, adenoacanthoma, miscellaneous), cellular atypsim (CAT I, II, III), structural atypism (SAT 1, 2, 3), infiltrative tendency (INF α, β, γ), grade of depth-invasion, lymphangitic and vascular invasion, and lymph node metastasis.

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Blood or blood components (plasma, leucocytes, and erythrocytes) were collected from normal rats as well as from rats with acquired resistance against tumor transplanted. The blood or components were transfused into rats via the tail vein before and after transplantation of tumor cells, and the survival time of the rats was observed. Ascites tumors used were Yoshida sarcoma and an ascites hepatoma, AH-712 cell line. The following results were obtained.
1) Blood of rats with acquired resistance against tumor transplantation was transfused into tumor-susceptible rats prior to inoculation with the same tumor. A striking anti-transplantation effect was found (prophylactic effect).
2) Not only the blood of resistant rats but also normal blood showed a prophylactic anti-transplantation effect.
3) The effect seemed to be due chiefly to leucocytes in the transfused blood.
4) The anti-transplantation effect of blood or blood component transfusion persisted for a few months.
5) Blood transfusion after tumor transplantation (therapeutic transfusion) was not so effective as prophylactic transfusion.

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The carcinogenic effects of tertiary-butyl hydroperoxide and t-butanol were tested to determine whether or not they could summate with the effect of 4-nitroquinoline 1-oxide to induce tumor in mice. Nine tumors were induced by t-butyl hydroperoxide and only 1 tumor by t-butanol when applied after submanifestational dose of 4-nitroquinoline 1-oxide. The difference between the number of tumors produced by these two compounds was statistically significant and the carcinogenicity of t-butyl hydroperoxide might be due mainly to oxygen-containing free radicals (RO•) produced by t-butyl hydroperoxide.

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Administration of heparin or dextran sulfate to rats prior to the intravenous injection of 3×106 transplantable rat lung carcinoma cells resulted in a significant decrease in pulmonary metastasis. Quantitatively, in the groups pretreated with heparin or dextran sulfate, the tumor area covered 4.9% or 10.4% of cross-sectioned surface of the lung while it was 56.0% in the control group. When time-lapse changes in the amount of intravenously injected ⁵¹Cr-labeled tumor cells in the lung were traced, it was found that tumor cells passed through the lung more rapidly and that more tumor cells were found in the circulating blood after pretreatment with heparin. These results seem to indicate that the tumor cells may be flushed away by heparin or dextran sulfate through the lung by blocking the formation of fibrin clots around the cells in capillaries resulting in a decrease of metastasis. From the result of preliminary studies on the cell electrophoresis, increased negative electric charge on the cell surface induced by heparin or dextran sulfate may also have contributed to the decreased mutual adhesiveness between capillary endothelium and tumor cells. Dextran sulfate may be applied clinically with minimal ill effect for the purpose of inhibiting metastasis of carcinoma.

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Chromosomes of golden hamster embryonic cells transformed to malignancy in vitro by treatment with 4-nitroquinoline 1-oxide and its derivative, 4-hydroxyaminoquinoline 1-oxide were examined with special regard to karyotype change in different stages of malignant transformation. In 5 among 11 transformed cell lines the chromosome numbers had a tetraploid mode, a diploid mode in 4, and in the remaining 2 the distribution was bimodal with a tetraploid and a diploid peak. One spontaneously transformed line showed a hyperdiploid mode. Generally, cells at an early stage of malignant transformation by treatment with the carcinogens had diploid or near-diploid chromosome number, but these at later stages after transformation had near-tetraploid chromosome numbers. Karyotypes of cells with diploid chromosome number, however, deviated more or less from those of normal somatic cells. The first event of cell transformation on chromosomal level seems to consist of structural chromosome changes, such as gaps, breaks, and deletion, whereupon heteroploid and polyploid karyotype changes follow due to non-disjunction and/or duplication of chromosome sets. Cells with such altered karyotypes might, when selected, establish malignant cell lines.

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Activation of cyclophosphamide took place not only in the liver of mice but also in the bone marrow of rabbits or in the kidney of mice and can thus be considered to be activated in all the organs in the body to a various extent. The activation was also demonstrated in human organs, such as the liver or bone marrow. Activated substance appeared in a short period. Solid Yoshida sarcoma or Ehrlich carcinoma activated cyclophosphamide but ascites form did not. This difference may be due to the presence of interstitial tissue of solid tumor. Various human carcinomas obtained at surgery activated cyclophosphamide in their solid form and, therefore, the administration of cyclophosphamide by means of regional perfusion might designate clinical significance. The action of bone marrow will also be helpful for the evaluation of its effect.

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The effect of continuous oral administration of 0.09, 0.04, and 0.025% 3-methoxy-and 2, 5-dimethoxy-4-aminoazobenzene (3-MeO- or 2, 5-(MeO)₂-AB) in the diet was studied using male Donryu rats, and following results were observed:
1) Liver tumors developed in the majority of rats given 3-MeO-AB.
2) More extrahepatic tumors were observed in the rats given 0.09% 3-MeO-AB than in those given 0.04% or 0.025% 3-MeO-AB. Among 26 animals receiving 0.09% 3-MeO-AB, tumors of the spleen and ear duct developed in 3 animals and tumors of the skin and small intestine in 2. With the lower concentrations of 3-MeO-AB, only 2 animals out of 46 developed tumors of the spleen.
3) 0.09% 2, 5-(MeO)₂-AB diet was very toxic and all 32 rats given this diet died during the 3rd week.
4) All the animals that received lower concentration of 2, 5-(MeO)₂-AB in the diet showed severe damage of the testicular tubules characterized by necrosis, calcium depostion, and arrest of spermatogenesis. Hypertrophy of the adrenal medulla was noted in a few animals.

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A new cell line derived from newborn Chinese hamster lung tissue was established in in vitro culture. This cell line (referred to as CHL) grew as monolayers with a doubling time of about 30 hours, showing fibroblastic appearances in morphology. Chromosome analysis of CHL line revealed that 54% of metaphases were normal female diploid. Inoculation of the cells at low densities less than 200 cells per dish gave rise to discrete colonies different in size and feature, with a plating efficiency of 16-20%.

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Total energies of alkylnitrosamines were calculated by means of an advanced molecular orbital method, in which all valence electrons were taken into consideration. Comparing the obtained energies for various possible stereo-structures, the most stable structure for methylethylnitrosamine was presumed. Hydroxylation at the α-carbon atom and heterolysis following it during metabolic conversions of nitroso compounds are readily explained from the obtained electronic charges. Assuming that alkylnitrosamines follow the currently accepted scheme of metabolic conversion toward the alkylation of guanine, energy increment, ΔE, involved in each step was calculated as the total energy of products minus the total energy of reactants. Similarly, ΔE was calculated for non-carcinogenic analogs assumed to follow the same metabolic pathway. Since it was found that the ΔE value for the carcinogen becomes smaller than that for the non-carcinogen only for the alkylation step occurring through bimolecular nucleophilic substitution, it was concluded that this alkylation step is the essential step involved in the assumed carcinogenic process of alkylnitrosamines.

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Enzymes were extracted from the liver of normal and Rhodamine sarcomabearing rats. The extracts were subjected to isoelectric fractionation with Ampholinecarrier ampholytes and divided into their pI-isozymes. Glutamate dehydrogenase from normal rats was mostly in a particle-bound form, and divided into at least four fractions of different isoelectric point (pI). Of these fractions, the fraction of pI 5.14 (pI 5.14-isozyme) was responsible for most of the total activity, and its activity was hardly influenced by the tumor. Besides the bound enzyme, there was a free enzyme, which was so labile as to be totally inactivated during isoelectric fractionation. The activity of the free enzyme was higher in tumor-bearing than normal rats.
The enzyme, 6-phosphogluconate dehydrogenase, was divided into pI 6.16- and pI 6.82-isozymes. Both pI-isozymes were hardly influenced by the tumor up to the middle stage of its growth. At the late stage, pI 6.16-isozyme decreased, but pI 6.82-isozyme remained unchanged.
Glucose-6-phosphate dehydrogenase was divided into pI 5.20- and pI 5.90-isozymes. The activity of the former was remarkably higher than the latter in normal rats. With growth of the tumor, pI 5.20-isozyme decreased and simultaneously pI 5.90-isozyme increased. This concurrent decrease-increase has been termed a "seesaw change." In most cases, the decrease and increase were comparable in extent; thus, the total activity of the two pI-isozymes was hardly changed by the tumor. Injection in normal rats of in vivo-liver enzyme-influencing substance prepared from the tumor16) caused the seesaw change, suggesting that the seesaw change in tumor-bearing rats was brought about by the substance produced in the tumor.
The two pI-isozymes of glucose-6-phosphate dehydrogenase showed a strict specificity on glucose 6-phosphate and NADP⁺; neither galactose 6-phosphate nor fructose 6-phosphate acted as a substrate, and NAD⁺ was not substituted for NADP⁺. The values of Km of pI 5.20-isozyme and pI 5.90-isozyme were 1.5×10^-5M and 6.9×10^-5M, respectively, for glucose 6-phosphate and 8.4×10^-6M and 1.2×10^-5M, respectively, for NADP⁺.

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Hepatoma 3683, an undifferentiated carcinoma of the liver, was transplanted subcutaneously. Animals with excision of both the transplant and regional lymph nodes had metastases to the lungs and thymic lymph nodes. Animals with excision of the transplant only had similar metastases, as well as metastases in the regional lymph nodes. Animals without surgery died without metastases.

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Chromosome aberrations such as chromatid gap, chromatid break, and chromatid exchange were induced in cultured Yoshida sarcoma cells by the minimal growth-inhibitory concentration (10^-8M) of 4-nitroquinoline 1-oxide. Persistent nucleoli in prometa- and meta-phases increased significantly by this carcinogen.

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