PRESENCE OF LARGE BENIGN CELLS IN T H E BLOOD CIRCULATION OF HAMSTERS WITH GRAFTED TUMORS

1 7 . TRAYKOVICH, M.D., 0. NGGEz-MONTIEI., M.D., M. R I V I ~ RM.D.,* E, M. GUBRIN, M.D.

AND

of patients suffering from malignant tumors, but they can be found only with great difficulty because of the presence of a vast number of large benign cells that are not normally present. These benign cells are from hematopoietic and connective tissues and are quite often erroneously diagnosed as cancer cells. Some authors have named them atypical cells,@abnormal cells,l~ 2 , 4 and large benign cells.7 T h e last expression is considered the most appropriate. During the studies carried out on grafted tumors in hamsters,g large benign cells, identical to those observed in human patients, were found in the circulating blood with the production of circulating cancer cells and metastasis. T h e morphology of such large benign cells in hamsters with different tumors is described in this paper.

UMOR CELLS CAN BE FOUND IN THE BLOOD

tion2 and then centrifuged again. Finally, smears were made with the sedimented cells and stained by the May-Grunwald-Giemsa, Papanicolaou, and Fontana methods. All of the animals were autopsied. The tissues were fixed in Bouins solution and stained with hematoxylin and eosin.

RESULTS

MATERIALS AND METHODS

Herbeuval et al.s3 modification of Traykovich et al.s technique9 was used to determine leukocyte concentration. T h e series consisted of 62 venous blood samples from 17 hamsters with melanoma, 11 with fusicellular sarcoma, 8 with endotheliosarcoma, and 26 control animals. T h e tumors were maintained by grafting. T h e blood samples were drawn by right heart puncture and were diluted in a phosphate-buffered solution with polyvinylpyrrolidone, sodium citrate, and 2% Formalin. A saponin solution containing 50% ethyl alcohol and 2% Formalin was then added by droplets until the erythrocytes were completely hemolyzed. T h e mixture was centrifuged at 2,500 rpm, and the sediment was washed with Herbeuval and Herbeuvals solu-

From the Instituto Venezolano de Investigaciones Cientificas, Apartado 1827, Caracas, Venezuela. * Present address: Institut de Recherches sur le Cancer, Villejuif, France. Received for publication June 19, 1964.

Of the animals with cancer, 64% showed large circulating nonmalignant cells, classified as originating from hematopoietic and connective tissues. T h e majority belonged to the megakaryocytic series and some were of the myeloid line. No large cells of the erythropoietic line were noted. On the other hand, 50% of the control animals presented circulating nonmalignant cells that were only mature megakaryocytes and endothelial cells. Among the hamsters with melanoma, 8 showed metastasis: 5 in the lung, 1 in the liver, and 2 in the lung and liver. Six of the 8 had immature cells; 5 of the 9 animals without metastasis also had immature cells. Four of the 11 animals with fusicellular sarcoma had metastasis in the lymphatic gland and showed circulating immature cells; 4 of the 7 animals without metastasis also had immature cells. I n the 8 hamsters with endotheliosarcoma, metastasis was seen in 6; 3 of the 6 had immature forms in the blood, and 1 of the 2 without metastasis also showed these immature forms. Megukuryocytic Line Cells. Normal (Fig. 1) and aberrant (Fig. 2) cells were found, as well as some mature cells (Fig. 5). T h e sizes varied from 50 to 100 p. T h e normal megakaryocytes were clearly identified because of their larger size. T h e aberrant cells showed irregularly shaped nuclei of wide variability and cytoplasmic tinctorial properties of the normal megakaryocytes (Fig. 2). T h e aberrant forms could be considered as originating by means of extramedullary hematopoiesis. Megakaryocytes were also seen in normal animals, but

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LARGE BENIGN CELLS IN BLOOD OF EXPERIMENTAL ANIMALS

Truykovich et ul.

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always in mature forms; they were easily identified because of the characteristic platelet formation (Fig. 3). Myeloid Line Cells. A myeloid reaction was observed in the blood of the hamsters with cancer, the lymphocytes having been replaced by polynuclear cells. The differential analysis of the circulating blood of normal hamsters showed 50 to 96% lymphocytes and 3 to 43% neutrophils. In these animals the differential

count was inverted. Moreover, immature elements, ranging from myeloblasts to metamyelocytes, were noticed; they showed the usual characteristics and always exceeded 15 p in size. Figure 4 shows a myelocyte, and Fig. 5 shows a metamyelocyte. Mononuclear Cells. Mononuclear cells were observed. They showed azurophilic granulation in the cytoplasm. The nuclei were more or less similar to those of the normal mono-

FIG. 1. A normal megakaryocyte with typical nuclei and abundant cytoplasm. (May-Grunwald-Giemsa. ~ 7 5 0 . ) FIG. 2. An aberrant megakaryocyte with very irregular and segmented nuclei from an animal with cancer. (May-Grunwald-Giemsa. x950.) FIG. 3. A megakaryocyte in the disintegration stage producing platelets. (May-Grunwald-Giemsa. X850.) FIG. 4. A myelocyte with a typical large nucleus and granular cytoplasm. (May-Grunwald-Giems. ~1,100.) FIG. 5. A metamyelocyte showing an unsegmented kidney-shaped nucleus displaced to the periphery with granulation in the cytoplasm. (May-Grunwald-Giemsa. ~1,600.) FIG. 6. A large mononuclear cell with azurophilic granulation. (May-Grunwald-Giemsa. ~950.) FIG. 7. Bare endothelial-cell nuclei. (May-Grunwald-Giems. ~1,5500.) FIG. 8. A typical mitosis in anaphase observed in control and cancer animals. (May-Griinwald-Giems. XI ,500.) FIG. 9. An atypical mitosis showing a roselike arrangement. (May-Grunwald-Giemsa. X1,150.)

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CANCER December 1964

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cyte. T h e mononuclear cells of animals with cancer sometimes appeared larger (18 to 25 p) than normal (Fig. 6 ) . Endothelial Cells. Endothelial cells were rarely found as single cells but were quite often seen in groups. Bare nuclei were more frequently observed than intact cells. T h e cytoplasm was stained pale blue and had round or elongated nuclei that showed granular chromatin. These cells belonged to large blood vessels and capillaries (Fig. 7). Mitotic Forms. Both typical (Fig. 8) and atypical (Fig. 9) mitotic forms were frequently observed. Romsdahl et al.5 reported the presence of mitotic cells in the blood of mice with experimental tumors. Cells in atypical mitosis showed irregular chromatin and polyploid forms, probably belonging to malignant cells. I n control animals, cells in mitosis were scarce. Undiagnosed Cells. Some cells could not be recognized, either because they had been damaged or because they were in a state of degeneration.

Herbeuval et al.3 also mentioned difficulty in identifying cancer cells in the blood of cancer patients, giving the same reason. Scheinin and Koivuniemi7 found these benign cells in the blood of 82y0 of patients suffering from cancer or certain noncancerous diseases and described the morphology of these cells. T h e significance of hematopoietic and tissue cells in the blood of tumor bearers is worth discussing. Extramedullary hematopoiesis in those with malignant tumors may be due to various causes, such as tumor infections, metabolic products, or toxins emanating from the malignant tissue, and metastasis in the bone marrow. I n addition, the myeloid reaction also occurs in the bone marrow of those with septicemia. Large benign cells are also seen in the blood of those with different types of anemia that are accompanied by extramedullary hematop o i e ~ i s .Therefore, ~ the hematopoietic reaction is easy to understand since the bone marrow is regarded as reacting to many abnormal conditions of the body as well as to malignant tumors.
SUMMARY

DISCUSSION
On the basis of the data presented, it has been proved that large benign cells found in the blood of hamsters with malignant tumors are similar to those found in human patients. These cells presented a problem in the detection of tumor cells i n the blood samples of laboratory animals grafted with tumors. On the basis of a relatively wide field of work, Sandberg et al.* concluded that, besides tumor cells, the blood of cancer patients contains large benign cells, difficult to identify. Bouvier et al.,1 Herbeuval and Herbeuval,2 and Large, uncommon benign cells were found to be present in the blood of hamsters grafted with metastasis-producing tumors. These hematopoietic and nonhematopoietic cells were found in 67% of the blood samples from laboratory animals with malignant tumors. Such cells were the cause of difficulties and errors in the diagnosis of malignant cells. These benign cells in the blood of experimental animals are similar to those found in human patients with cancer and other diseases.

REFERENCES

P. A., and ROCHE, R.: 1. BOUVIER, C. A.; MAURICE, Nature des cellules anormales trouvees dans le sang circulant en prbence daffections tumorales. Oncologia 15: 129-139, 1962. R., and HERBEUVAL, H.: La concen2. HERBEUVAL, tration leucocytaire; premiers rtsultats dans la recherche des cellules anormales. Compt. rend. SOC. biol. 154: 1261-1264, 1960. 3. HERBEUVAL, H.; HERBEUVAL, R.; CUNY,G.; DuHEILLE, J., and GUERCI, 0.: Etude du passage des cellules hematopoiktiques dans le sang circulant au cours des cancers; importance de la m6gacaryocytPmie. Nouvelle rev. franG. hemat. 2: 619-621, 1962. 4. HERBEUVAL, H.; HERBEUVAL, R., and DUHEILLE, J.: Le passage, dans le sang circulant, des amas hbmatopoiktiques, ilots Prythroblastiques, ilots megacaryoblastiques, ilots mixtes. Nouvelle rev. frunG. hemat. 2: 616-619, 1962. 5. ROMSDAHL, M. D.; CHU, E. W.; HUME,R., and

SMITH, R. R.: Time of metastasis and release of circulating tumor cells as determined in experimental system. Cancer 14: 883-888, 1961. 6. SANDBERC, A. A.; MOORE, G. E., and SCHUBARG, J.. R.: Atypical cells in blood of cancer patientsdifferentiation from tumor cells. J. Nut. Cancer Inst. 22: 555-565, 1959. 7. SCHEININ, M. T., and KOIVUNIEMI, A. P.: Large benign cells in circulating blood and their significance in identification of cancer cells. Cancer 15: 972-977, 1962. 8. TRAYKOVICH, V.; N~~Ez-MONTIEL, 0.; RIVIBRE, M. R., and GuDRIN,M.: Eine einfache Methode fur die Untersuchung der Krebszellen im Blut von Versuchstieren. Frankfurt. Ztschr. Path. 74: 1-3, 1964. 9. TRAJKOVIC, V. [TRAYKOVICH, V.]; RIVIBRE, M. R., and GuDRIN,M.: Recherche des cellules tumorales dans le sang chez le hamster porteur de diverses tumeurs. Bull. Assoc. franC. etude cancer 50: 263-274, 1963.