Selection of liver-colonizing tumor cells from a murine fibrosarcoma induced by methylcholanthrene.

An experimental tumor model was developed to study the organ preference of malignant tumors. The primary tumor ER 15-P was induced by inoculation of 1 mg methylcholanthrene in 0.1 ml sesame oil into the left femoral muscle of a female C57/Bl6J mouse. The tumor was palpable 100 days after induction. Spontaneous lung metastases were found at autopsy on day 128. Serial IM transplantation of tumor cells from the primary ER 15-P resulted in pulmonary metastases in all male and female mice. After IV injection of tumor cells from ER 15-P to male mice, colonies were found in lungs, thoracic cavity, liver, kidneys and occasionally also adrenals; female mice sometimes had ovarian metastases in addition, but no hepatic metastases. Liver-colonizing tumor cells were selected in male mice as follows: (a) IV injection of tumor cells from primary ER 15-P; (b) removal of tumor cells from liver tumor nodules, reinjection into mesenteric vein; (c) preparation of resulting tumors in the liver, reinjection of these cells through the portal system in one group of mice, and IV administration into tail vein in another group: (d) IM inoculation of tumor cells of the mesenteric passage in the left gastrocnemius muscle of mice prior to IV injection via tail vein in another group. Steps c and d were repeated three times. The procedure resulted in a highly significant decrease of tumor cell colonization to lungs and other organs, and a preferential increase of liver colonization. The liver preference of cell lines thus selected was obvious. Possible mechanisms for the organ preference of malignant tumors are discussed.

Histological grading and clinical staging of plasmacytoma.

One hundred and forty cases of myeloma were reexamined histologically and classified in three histocytological grades. Close correlations were established between the clinical stages as defined by Durie and Salmon (1975) and the grades of histocytological differentiation. The number of osteolytic lesions correlates with dedifferentiation of malignant plasma cells. Dedifferentiation and high plasma cell counts in the bone marrow are associated with rarefication of hemopoiectic cells and often with an increase of reticular fiber network.

Macrophages and T lymphocytes infiltrating the rat mammary carcinoma HH9-cl 14 in progressive and regressive tumor growth. An immunohistological study.

Ascites tumor cells (2 X 10(6] of a DMBA-induced rat mammary adenocarcinoma (HH9-cl 14) were injected s.c. into tumor-free syngeneic female rats and produced a continuously growing solid tumor in all animals of this group. Inoculation of 2 X 10(7) cells induced a first brief period of tumor growth, followed by complete tumor regression from the 2nd until the 5th week after injection. Both the progressive and the regressive tumors were analyzed immunohistologically at different stages with monoclonal antibodies against different T lymphocytes and macrophages. Obviously these cells appear in different quantity and quality, during the hosts immune response. Possible interactions of T lymphocytes and macrophages with tumor cells are discussed.

Flow cytometric DNA analysis of malignant lymphomas with primary bone manifestation.

Malignant lymphomas with primary skeletal manifestation have received controversial evaluation with regard to histological classification and histogenesis. Recent histological and immunohistological studies on the rare bone lymphomas conducted by our team, have shown that they do not differ from primary nodal lymphomas with regard to the spectrum of histological subtypes. The present flow cytometric DNA analysis of paraffin-embedded material from 17 lymphomas documented in the Bone Tumor Registry of Westfalia yielded the following distribution pattern of DNA ploidy: among 12 non-Hodgkin's lymphomas (NHL) (according to the Kiel classification) there was only 1 case of low grade malignancy; this centroblastic-centrocytic lymphoma showed a unimodal diploid DNA histogram. Of 11 highly malignant NHL, 6 were DNA hyperdiploid. Among the 5 cases of Hodgkin's lymphoma, 4 were DNA diploid, (1 nodular sclerosing, 3 mixed types) and one DNA tetraploid (lymphocytic depletion type). Comparison with data from the literature reveals that even with regard to DNA ploidy, malignant lymphomas primarily manifesting in bone do not differ from those of exclusively nodal manifestation.

Ultrastructure of telangiectatic osteosarcoma.

Recent investigations have shown that telangiectatic osteosarcoma has a poorer prognosis than other osteosarcomas. To elucidate the histogenesis of TOS two cases were investigated on the electron microscopic level. The results show that besides anaplastic, osteoblast-like, and fibroblast-like tumor cells angiosarcomatous components can be observed in this malignant bone tumor, which are characterized by endothelial cells with pinocytotic vesicles, tight intercellular junctions, fine fibrils, and so-called Weibel-Palade bodies in their cytoplasm. From these results, it is concluded that telangiectatic osteosarcoma is derived from multipotent mesenchymal cells with potential differentiation into various directions, such as osteoblast-like cells, and fibroblast-like cells.

Adenocarcinomas of esophagus and cardia in comparison with gastric carcinoma.

Since the carcinomas of the cardia and the adenocarcinomas of the esophagus show many similarities in their histological and morphological descriptions, a detailed comparative study was attempted on the basis of 66 esophageal carcinomas in adenoid differentiation, 359 carcinomas of the cardia, 1288 gastric carcinomas in infracardial localisation, and 492 squamous carcinomas of the esophagus. The evaluation yielded no significant differences between the adenocarcinomas of the esophagus and the cardia neither in age and sex distribution nor with regard to the classifications of Borrmann, WHO, Ming, and Laurén, but a significant discrimination was possible between esophageal and cardial adenocarcinoma together, on the one hand, and infracardial gastric carcinoma on the other. Furthermore, esophageal adenocarcinomas were localized preferentially in the lower third, unlike squamous carcinomas of the same organ. These results suggest that esophageal adenocarcinoma and carcinoma of the cardia must be considered as one separate entity, probably originating from a common stem cell. They further suggest that the cardia belongs to the esophagus rather than to the stomach.

Biologic characterization of human bone tumors. I. Ewing's sarcoma. A comparative electron and immunofluorescence microscopic study.

Six cases of Ewing's sarcoma were investigated by electron and immunofluorescence microscopy. A layer of basement membrane-like deposits was found between typical principal and secondary tumor cells. To clarify the nature of these ultrastructural deposits, antibodies against collagen type IV were applied to frozen sections of corresponding tumor tissue. This reaction revealed type IV collagen as a regular component of basement membranes in nonneoplastic tumor capillaries, but it was equally able to localize collagen type IV between single tumor cells in capillary-free areas. With the same method, factor-VIII-associated protein, predominantly found in endothelial cells, could be demonstrated in some tumor cells. These results demonstrate that, in addition to anaplastic cells, some tumor cells are found in Ewing's sarcoma that share certain differentiating features with the endothelial cell.

Benign fibrous histiocytoma of bone. Light- and electron-microscopic observations.

Case report of a patient with an unusual, rapidly growing bone tumor in the third and fourth cervical vertebrae. Histological and electron-microscopic investigations reveal a tumor composed of histiocytic cells, xanthomatous cells, giant cells of Touton type, and fibroblastic cells. No cellular features of malignancy are observed. From its cytologic appearance the tumor has to be classified as a benign fibrous histiocytoma. Regarding its ultrastructural features, the tumor may not be distinguished from non-ossifying fibroma of bone, but its clinical pattern shows obvious differences of localization and growth potential. the term "benign fibrous histiocytoma of bone" is proposed for these tumors which must be differentiated from non-ossifying fibroma.

Morphology of pulmonary metastases from osteosarcoma during chemotherapy.

Osteosarcoma is known to metastasize rather early, and even after surgical resection of the primary metastases may occur predominantly in the lung. Administration of polychemotherapy for destruction of micrometastases has served to improve prognosis. Preoperative chemotherapy facilitates the evaluation of regression, another factor of high prognostic relevance. Morphologic analysis of pulmonary metastases developing during chemotherapy is of considerable interest on account of the potential therapy resistance of certain histologic subtypes of osteosarcoma. In the present study pulmonary metastases resected in 20 thoracotomies of 15 osteosarcoma patients were investigated by light microscopy and compared, if possible, to the respective primaries. All patients had received chemotherapy, predominantly according to the COSS 80 and COSS 82 protocols. The histologic picture of a tumor was found to change from the primary to the pulmonary metastasis, a pattern also verified in the lung metastases collected in consecutive thoracotomies from the same patient. Several different subtypes were regularly found side by side in the metastases, but generally no special sensitivity or resistance to chemotherapy could be attributed to any of these subtypes. Our results nevertheless do indicate an increased resistance of anaplastic tumor tissue. The response to chemotherapy agreed in 9 of 10 primaries with that of their metastases.

Cytostatic effects in osteosarcomas as detected by flow cytometric DNA analysis after preoperative chemotherapy according to the COSS 80/82 protocol.

A total of 20 highly malignant osteosarcomas were studied by DNA flow cytometry after preoperative chemotherapy according to the COSS 80/82 protocol to assess their nuclear DNA content and the impact of chemotherapy on DNA ploidy and proliferation. Of the cases studied, 70% revealed aneuploid DNA stem lines with a median value of 1.67, and a median S-phase proportion of 16.1%. These data differed substantially from the results of a preceding study on untreated osteosarcomas revealing a higher frequency of DNA aneuploidy, higher DNA indices, and higher proportions of cells in S-phase. The pretreated tumors also showed a distinct relation between DNA content and the grade of tumor regression. No aneuploid DNA stem line was found in the responder group-I, whereas group-V (osteosarcomas with more than 50% viable tumor tissue) consisted almost completely of aneuploid DNA populations; all tumors with more than 2 DNA aneuploids were found in the latter group. All populations with a DNA index (DI) over 2.0 were found in the nonresponder groups IV and V, where all DNA aneuploids had an S-phase above 12%. No differences in DNA ploidy or proliferation were found in the various histological subtypes of pretreated osteosarcomas. These data indicate that flow cytometric DNA measurement in osteosarcoma may not only serve as a tumor marker, but also support the evaluation of morphologically established regression grades, thereby verifying the patient's prognosis: a high DI (over 2.0), a high number of DNA aneuploids (more than 2), and a high proliferation (S-phase above 12%) seemed to indicate a poor regression of the primary lesion, which may offer a pretherapeutic way of prognostic evaluation.

Biological characterization of human bone tumors. VIII. Expression of HLA-DR antigens in bone tumors and tumor-like lesions.

A total of 45 cases of bone tumors and tumor-like lesions were studied in order to determine the expression of an HLA-DR antigen by the monoclonal antibody 910-D-7, and its possible correlation with histology, using the indirect immunoperoxidase method on frozen sections. The pattern of antigen expression was nearly constant for the individual cell types, though varying in intensity, and did not depend on the biological behavior of the respective lesions. No clear correlation could be established between antigen expression and cell maturation. Although the biological significance of antigen expression in these tumors is not yet understood, it is clear that here, too, the mere presence of an HLA-DR antigen cannot be interpreted as a sign of malignant transformation.

Combined ultrastructural, histochemical, and autoradiographic study of osteosarcoma after preoperative chemotherapy according to the COSS 80 protocol.

Twelve osteosarcomas treated according to the COSS 80 protocol (preoperative chemotherapy, resection) were studied by light and electron microscopic, histochemical, and autoradiographic methods. Evidence of regressive and necrotic changes was found in many tumor cells, but the alterations were unspecific. Viable tumor cells of high malignancy were also observed regularly, often at the S phase. As the tumor regression continued, a strong reaction of the mononuclear phagocyte system was manifested by the presence of macrophages and giant cells.

Hepatic failure in a patient treated with dacarbazine (DTIC) for malignant melanoma.

A 55-year-old white woman received chemotherapy with DTIC after surgery for malignant melanoma (stage I, SSM IV, depth of invasion 12 mm). She died suddenly during the second treatment cycle. Autopsy revealed massive necrosis of the liver and thrombosis of the hepatic veins. The cause of the fatal outcome is attributed to the adverse toxic effects of DTIC.

Biological characterization of human bone tumors. X. The proliferation behavior of macrophages as compared to fibroblastic cells in malignant fibrous histiocytoma and giant cell tumor of bone.

Seven giant cell tumors of bone and four malignant fibrous histiocytomas were studied immunohistochemically with different monoclonal antibodies to the mononuclear phagocyte system (MPS), to HLA-DR antigens, and to a proliferation-associated nuclear antigen (KI-67), in order to clarify the role of macrophages in these tumors. A part of the mononuclear cells stained positive with antibodies against the MPS. Antibody 25-F-9 against mature tissue macrophages showed the strongest reaction. The osteoclast-like giant cells also stained positive with this antibody. Fibroblast-like stromal cells, however, showed negative reactions to all antibodies against MPS cells. A double-labeling immunohistological technique was used to detect the proliferating cell population in these tumors. The fibroblast-like cells that were negative for MPS markers, were positively labeled with the monoclonal antibody Ki-67 against a proliferation-associated nuclear antigen, whereas a negative reaction to Ki-67 was seen in cells positive with antibodies to the MPS. These results support the concept that macrophages are a reactive population in these tumors, whereas the fibroblast-like mesenchymal cells are the proliferating tumor cells.

Improved grading of bone tumors with the monoclonal antibody Ki-67.

A total of 60 bone tumors and tumor-like lesions presenting various grades of malignancy were investigated immunohistologically with the monoclonal antibody Ki-67 directed against a cell proliferation-associated nuclear antigen. The results obtained agree well with those of flow cytometric and autoradiographic studies on similar tumor entities. The monoclonal antibody Ki-67 was found to be a handy and reliable tool for improved grading of bone tumors.

Metastatic patterns of renal carcinoma: an analysis of 687 necropsies.

The metastatic behaviour of renal cell carcinoma has been studied in a series of 687 necropsies. The observations were consistent with the concept of "metastatic inefficiency", in that in 295 cases, including 25 with renal vein invasion, there were no detectable metastases. In the present series, renal vein involvement was not an important prognostic factor in stage 1 or 2 disease. In 73% of cases without lung metastases there were none in other sites, and in 84% of those with lung metastases there were others elsewhere, consistent with a metastatic "cascade" in which metastases first developed in the lungs and were later detected in other organs. However, the observations did not permit discrimination between anatomic cascades, in which other organs were seeded from metastasizing pulmonary metastases, and temporal cascades, in which the other were seeded at the same time as the lungs, but with fewer cancer cells. The patterns of arterial metastasis were consistent with the "seed-and-soil" hypothesis, and a novel index was developed to quantify differential organ "soils". The contralateral kidney was not the best soil for metastases from renal carcinoma. Given the presence of lymph node metastasis, the probability of heamatogenous metastasis is 90%. However, in the absence of nodal metastasis, approximately half the cases had haematogenous metastasis.

Precancerous conditions and epithelial dysplasia in the stomach.

A distinction can be made between a precancerous condition and a precancerous lesion. The former is a clinical state associated with a significantly increased risk of cancer, whereas a precancerous lesion is a histopathological abnormality in which cancer is more likely to occur than in its apparently normal counterpart. Up to the present time atrophic gastritis, gastric ulcer, pernicious anaemia, gastric stumps, gastric polyps, and Ménétrier's disease have all been considered as precancerous conditions and lesions of the stomach. Of these, only atrophic gastritis, pernicious anaemia, gastric stumps, and certain types of gastric polyp can now be regarded as having any really significant malignant potential. The precancerous lesion common to these is epithelial dysplasia which can occur in ordinary (foveolar) gastric epithelium as well as in intestinal metaplasia. The criteria for grading dysplasia in gastric epithelium into mild, moderate, and severe grades are given, and attention is drawn to the problems of differentiating inflammatory or regenerative change from mild dysplasia and intramucosal carcinoma from severe dysplasia. The clinical and epidemiological implications of gastric dysplasia are discussed with suggestions for further research.

Decreased sensitivity of isolated hepatocytes from baby rats, from regenerating and from poisoned livers to phalloidin.

Isolated hepatocytes, prepared from 5 day old rats, from regenerating livers of from livers after poisoning with carbon tetrachloride, are less sensitive to phalloidin in vitro than hepatocytes from untreated adult controls. The time course of the reduced susceptibility to phalloidin was compared with the ability of hepatocytes to take up bile acids under various conditions. SDS-electrophoresis of cell lysates gave no evidence for decreased levels of actin in cells with reduced sensitivity to phalloidin. In contrast, there was a good relationship between the active uptake of bile acids and the sensitivity of hepatocytes to phalloidin. The decreased response of hepatocytes from baby rats, from regenerating livers or from poisoned livers to phalloidin is more probably related to differences in phalloidin uptake than to a reduced endowment with microfilamentous structures.

Inhibitory effects of 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid (DIDS) in the response of isolated hepatocytes to phalloidin.

4,4'-Diisothiocyano stilbene-2,2'-disulfonic acid (DIDS) inhibits the typical development of protrusions, regularly seen after treatment of isolated hepatocytes with phalloidin. The degree of inhibition depends on the time of preincubation and on the concentration of DIDS, but not on the concentration of phalloidin. DIDS is more effective than H2DIDS. The inhibition by both compounds is irreversible. The binding capacity of hepatocytes for H2DIDS is much higher than that of the phalloidin-insensitive hepatoma cells. Gel electrophoresis of lysates from cells, pretreated with 3H2DIDS demonstrates that actin binds very little of the inhibitor. Our results suggest that a protein structure on the surface of hepatocytes, needed for the response to phalloidin, is influenced by DIDS or H2DIDS.

A possible reason for the phalloidin tolerance of hepatoma cells.

In contrast to normal liver cells, AS-30D hepatoma cells are insensitive to phalloidin. The lack of the typical phalloidin response in the latter cells is not due to a deficiency of contractile proteins. Actin prepared from hepatoma cells is able to form filamentous structures and is stabilized in a manner similar to muscle actin. Isolated liver cells were exposed to a medium containing phalloidin and removed after 20 min by centrifugation. The supernatant was incubated again with fresh cells. The procedure was repeated four times. The phalloidin response decreased to about 19% of the control because of the uptake of phalloidin during each incubation. When the same procedure was carried out with AS-30D hepatoma cells, and aliquots of the supernatants were tested with hepatocytes no marked decrease of the phalloidin response was seen. This indicates that hepatoma cells do not consume the toxin as do normal liver cells.