The Committee on International Collaborative Activities (CICA) of the International Union Against Cancer (UICC) has decided to attempt to provide a current international standardized terminology in cancer (tumor) etiology, in cooperation with the International Agency for Research on Cancer (IARC) and the World Health Organization (WHO). As a first stage of this attempt and to encourage international discussion, CICA has requested one of its members, the author, to formulate and publish under his responsibility the present analysis and proposals. The present paper to be published in a number of cancer journals outdates all of its previous versions or drafts.

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A permanent cell line (HLC-1) was established from the pleural effusion of a human lung adenocarcinoma. The cell line was characterized by the monolayered and multilayered organoid growth of epithelioid cells with the doubling time of about 33hr and the modal chromosome number of 68. Cloning efficiency was 17.9% in liquid medium and 8.3% in soft agar. The cells produced a large amount of epithelial mucin. Electron microscopic examination revealed many secretory granules and terminal bars. They formed spherical aggregates in a gyratory culture which showed adenocarcinoma-like tubular structures histologically. Enzyme-histochemically, they showed the characteristics of lung adenocarcinoma cells except for a few enzymes such as glucose-6-phosphatase and ATPase. Heterotransplantation of the cells produced the tumor. These characteristics confirm that HLC-1 cell line is a human lung adenocarcinoma cell line.

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HeLa cells were heterotransplanted to nude mice and their response to some of antitumor agents was investigated. HeLa cell-tumor readily grew in nude mice and no regression was observed. Metastases to the lung and other organs were noticed in some of the animals. Histopathological examination revealed that the tumor retained the original characteristics of human epidermoid carcinoma.
Standardization of HeLa cell-tumor in nude mice for the screening and evaluation of antitumor chemotherapeutics was attempted. Marked inhibition of tumor growth was observed with lower doses of Mitomycin-C, 5-fluorouracil, Adriamycin, and Bleomycin. The tumor regression was observed with high doses of Mitomycin-C, Adriamycin, and 5-fluorouracil. However, cyclophosphamide, cytosine arabinoside, and daunorubicin had little effect on the tumor growth. Complete regression was not obtained with any of the test agents and active regrowth took place even with the most effective compound. Considerable variation in the effect on tumor growth was observed among the test compounds, while histopathological findings were much alike; few mitotic figures, vacuolization, and pyknosis were main changes in tumor cells, and large foci of necrosis and hemorrhage were present in the degenerative areas. The regrowth was initiated around the capillaries in the necrotic tumor tissue.

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Cytotoxicity of peripheral blood lymphocytes against a cultured cell line QG-56 of a bronchogenic squamous cell carcinoma was studied by a microcytotoxicity test in 33 cases of bronchogenic carcinoma. An augmented cytotoxicity was detected in the patients in early stages of the disease as compared to normal controls or the patients carrying other types of tumors. The cytotoxicity declined as the stages of cancer advanced. The patients with high degree of cytotoxicity appeared to show better clinical course after radiotherapy.

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A transplantable osteogenic sarcoma originally arising in the right femur of an AKR/Ms male mouse is described. The original tumor showed a conspicuous bone and cartilage formation, but the capability of forming the bone was lost in the 4th transplant generation and that of cartilage formation was lost in the 7th generation. Alkaline phosphatase activity was histochemically demonstrated in only a few tumor cells, and the activity did not rise in the host serum. Intracis- ternal type-A particles of an average diameter of 70nm were abundant in the rough endoplasmic reticulum, while type-C particles were rarely found to be budding from the cell surface or free in the extracellular spaces by electron microscopy. Three of the 27 thymectomized AKR mice that had been neonatally injected with cell-free material of the transplanted tumor developed osteomas, but no osteogenic sarcoma was found.

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Several anticancer chemicals containing a quinone group were found to stimulate the aerobic oxidation of NADPH by liver microsomes. The enzyme responsible for the above reaction was identified as NADPH-cytochrome c reductase (EC 1.6.2.4), one of the microsomal flavoproteins. The fact that a catalytic amount (20μM) of these anticancer chemicals was sufficient to oxidize all the NADPH (100μM) indicates that they function as electron carriers from the flavopmtein to molecular oxygen. As a corollary, Mitomycin-C and Carbazilquinone stimulated oxygen uptake by Ehrlich ascites tumor cells in the presence of glucose but Daunomycin and Adriamycin failed to do so, although the reason for it remains to be elucidated. Carbazilquinone, in contrast to others, also stimulated the microsomal NADH oxidation.

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The effect of a low molecular weight sulfated polygalacturonic acid on the growth of BALB/c 3T3 cells and HeLa cells was examined. This chemical reduced the saturation density of 3T3 cells in the range of concentrations of 10^-4 to 1mg/ml, and inhibited the proliferation of HeLa cells, causing little or no cell damage. Treated cells began to grow after removal of the sulfated polygalacturonic acid. Treatment with this acid also induced enlargement of the cell size in 3T3 cells, and a marked reduction of colony size and of central piling up in HeLa cells. ³⁵S-sulfated polygalacturonic acid was bound to the cells immediately. Electrophoretic mobility of both cell lines treated with this sulfated polygalacturonic acid increased the negative charge of the cell surface. These evidences suggest that sulfated polygalacturonic acid affects the cell surface, restricting the cell growth of both cell lines.

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Sixty patients with advanced gastric cancer were subjected to the macrophage electrophoretic mobility test and delayed hypersensitivity skin test to tumor and normal tissue (gastric mucosa) extracts. Tumor extract induced significant inhibition of macrophage mobility in 51% and positive skin reaction in 32% of examined cases. Close correlation was observed between macrophage inhibition and diameter of skin reaction to tumor extract. Incidence of positive reaction in both tests to normal tissue extract was 55% and 12%, respectively. Significant inhibition of macrophage to tumor extract was also observed in breast cancer, but not in superficial gastric cancer and healthy persons. Normal tissue extract did not induce significant inhibition of macrophage in gastric ulcer. These results suggest that macrophage electrophoretic mobility test indicates a certain aspect of tumor-associated (but not tumor-specific) immunity in cancer patients. Macrophage inhibiton appeared to be related with macroscopic tumor types rather than to clinical stages. Positive skin reaction to tumor extract was observed in Stages II, III, and IV of gastric cancer.

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Three groups of ACI/N rats of both sexes were given 400, 200, or 100ppm of N-propyl-N-nitrosourethan (PNUR) continuously in the drinking water. The incidence of tumors in the upper digestive tract was 100% in all 3 groups. These tumors were observed most frequently in the forestomach, followed by the esophagus, oral cavity, and pharynx. Histologically, all the tumors were papillomas or squamous cell carcinomas A few tumors were detected in the small intestine and glandular stomach.
The study on morphogenesis of squamous cell carcinomas arising from the upper digestive tract indicates that the majority of tumors of the esophagus and forestomach may pass through acanthosis or hyperkeratosis, leukoplakia, leukokeratosis, and papilloma, and finally develop into invasive squamous cell carcinoma, but many carcinomas of the oral cavity and pharynx, especially the tongue, may develop without passing through a papillomatous stage.

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Thymidine kinase was partially purified from human adrenocortical carcinoma, hyperplasia, and normal adrenal glands. For the purpose of clarifying the qualitative and quantitative difference of thymidine kinase between cancer and normal tissue, biochemical properties of partially purified thymidine kinase were compared. Adrenocortical carcinoma and hyperplasia contained greater concentration of thymidine kinase than normal adrenal gland. By the DEAE-cellulose chromatography, adrenocortical carcinoma gave two peaks (Peak I and Peak II) of thymidine kinase, while in hyperplasia and normal adrenal gland, the second peak (Peak II) was only slightly detected or hardly detected. Thymidine kinase in these three glands was identical with respect to pH optimum and inhibition by dTTP, but inhibition by dCTP was quite different. dCTP inhibited the activity of normal adrenal gland and Peak II of adrenocortical carcinoma by 55% and 40% at 0.1mM, respectively, but the activity of adrenocortical hyperplasia and Peak I of adrenocortical carcinoma was hardly affected. Normal adrenal enzyme was more stable against heat inactivation than adrenocortical carcinoma and hyperplasia. The apparent K_m with thymidine for Peak I and Peak II of adrenocortical carcinoma, hyperplasia, and normal adrenal gland was 5.0, 11.1, 5.1, and 25.0×10^-6M, respectively.

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DL-α-Hydrazino-δ-aminovaleric acid (DL-HAVA) is a potent and fairly specific inhibitor of ornithine decarboxylase (EC 4.1.1.17). Its effect on polyamine metabolism and cell proliferation was investigated in sarcoma-180, inoculated into the axillary region of mice. In the tumor tissues, the activities of ornithine and S-adenosyl-L-methionine decarboxylases and the putrescine level were much higher in the early stage of growth than those in normal mouse liver. Administration of DL-HAVA greatly depressed the putrescine level and putrescine formation from L-ornithine. It also suppressed DNA synthesis and increase in weight of the tumor tissue. However, it had little effect on RNA synthesis or the tissue concentration of spermidine and spermine. The inhibition of DNA synthesis and subsequent tumor development by DL-HAVA was effectively prevented by putrescine, but not by cadaverine or 1, 7-diaminoheptane.
From these results it is concluded that the suppression of DNA synthesis and neoplastic growth by DL-HAVA is due to decrease in the putrescine level by inhibition of ornithine decarboxylase.

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The kinetics of radiation-induced cell death was compared in in vitro and in vivo systems, using mouse mammary carcinoma cell line, FM3A. Cultured and ascites cells were irradiated with 5 krad of X-rays, and changes in then umber of viable cells and in the cell volume after irradiation were analyzed. The volume of irradiated cells in both systems increased about 5 times that of control after 3 generations and was saturated. Cells in vitro began to die after 3 generations. The number of cells in vivo began to decrease immediately after irradiation, but the rate of the decrease became large at 3rd generation. It was concluded that radiation-induced lethal damage manifests itself in the same way at the intracellular level in both systems, and some dying cells in vivo were eliminated before manifestation of lethal damage.

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The hepatocellular mitotic response associated with Ehrlich ascites tumour is mediated by the tumour less than 48hr, but more than 18hr prior to the time of observation (day 8 of tumour growth).
Tumour-associated hepatocellular mitosis is normally centrilobular in distribution and appears to involve marginally more of the liver lobule than is sensitive to the necrotic effect of CCl₄.
There is failure to summate mitotic activity when both tumour-associated hepatocellular mitosis and regenerative hepatocyte mitosis would be expected to occur; in this situation regenerative hepatocyte mitosis is the overwhelming contributor to the observed mitotic index. It is postulated that tumour-associated hepatocellular mitosis is obligatorily centrilobular.
Tumour-associated hepatocellular mitosis, on the evidence of the present work and the results of others using intraperitoneally administered proteolytic enzymes as inducers of hepatocyte mitosis, is considered to be systemically mediated; there is complete failure to observe any perilobar preponderance of mitotic activity. This implies an indirect effect of tumour in producing the hepatocyte mitogen.
Criteria obtained from the present work are suggested as useful in any attempt to isolate and identify the true or in vivo tumour-associated mitogen, and would help to exclude possible non-specific mitogens.

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In order to clarify the mechanism of potentiation of cyclophosphamide activity by centrophenoxine, blood concentration of total and activated cyclophosphamide was examined. Blood concentration of cyclophosphamide increased by the compound and biological half-life of activated cyclophosphamide was markedly increased to 30min from 18min in intraperitoneal administration. At the same time, concentration of active form in ascites fluid was also increased and biological half-life of the active form was increased to 50min from 18min. Similar increase in blood level of active form was also shown by p-chlorophenoxyacetic acid and probenecid, but concentration at early stages after injection was not increased. As a result, potentiation of cyclophosphamide activity by centrophenoxine was found to be due to maintenance of active form in both blood and ascites fluid at higher levels than those in the control.

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A potent immunosuppressive principle was isloated in a soluble form from the nuclei of Ehrlich ascites tumor cells. DNA-free preparation, obtained from the purified nuclei by homogenizing in 2M NaCl-5M urea solution and removing the dissociated DNA with LaCl₃-precipitation, revealed strong activity to suppress the development of helper thymus-derived lymphocyte activity in mice. Moreover, almost all of the immunosuppressive activity originally found in the nuclear residue fraction after extraction with sodium citrate solution was recovered in 0.25N HCl extract which mainly contains proteins. The nuclear components of tumor cells were also detected in the body fluid of tumor-bearing animals, as examined by the reactivity with a rabbit antiserum specific for some nuclear component of Ehrlich tumor cells. These results suggest that the immunosuppressive principle in the nuclei of Ehrlich tumor cells is a histone-like substance, and liberated into the body fluid from tumor cell nuclei in the tumorbearing state.

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A synthetic stearoylmannan phosphate, designated as SMP-Fr. I-d, consisting of three components, fatty acid, polysaccharide, and phosphate, enhanced the therapeutic effect of Mitomycin-C in mice implanted with 1×10⁷ cells of Ehrlich carcinoma ascites form. Namely, 50% of the mice survived more than 60 days when ineffective doses of 0.5mg/kg of Mitomycin-C and 50mg/kg of SMP-Fr. I-d were administered intraperitoneally once daily for 7 days. SMP-Fr. I-d was found to be virtually non-toxic even in a dose of 1, 000mg/kg intraperitoneally in mice.

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An experimental cancer induction in subcutaneously autoimplanted bronchial grafts in dogs was studied. After successful bronchial autografting, 3-methylcholanthrene or 3, 4-benzo[a]pyrene suspended in sterile saline solution containing 1% carboxymethylcellulose was injected into the lumen. Twenty-eight weeks after this treatment, an invasive squamous cell carcinoma with concomitantly existing atypical squamous metaplasia was found in one dog by histological examination. Squamous cell metaplasia was also observed in two other dogs at 24th experimental week. Cytological examination revealed the findings corresponding to these epithelial changes. Remaining 15 dogs are still living and under observation.

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A simple, sensitive, and convenient method combining thin-layer chromatography and a spot test for mutagenicity of Salmonella typhimurium TA100 was utilized for the analysis of urine of rats fed N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT), a potent experimental urinary bladder carcinogen. Three metabolites of FANFT were detected in urine, and one of these, accounting for 33% of the urinary metabolites of FANFT, was identified as 2-amino-4-(5-nitro-2-furyl) thiazole (ANFT). ANFT was the only urinary metabolite that clearly demonstrated mutagenic activity, suggesting that ANFT may be a proximate vesical carcinogen of FANFT.

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A significant correlation between phytohemagglutinin (PHA) and delayed cutaneous hypersensitivity to dinitrochlorobenzene was obtained in 82.2% of 45 cancer patients. PHA skin test seemed valuable for a serial evaluation of immunocompetence in cancer patients.

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Asterriquinone isolated from the mycelium of Aspergillus terreus was tested for its antitumor activity. This compound was very effective against Ehrlich carcinoma and ascites hepatoma, AH-13, in vivo, while slightly effective against Yoshida sarcoma and L-1210 leukemia. Some modifications in the molecule resulted in entire loss of its activity.

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