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PRESENCE OF LARGE BENIGN CELLS IN THE BLOODC I R C U L A T I O N OF H AM ST E R S W I T H G R A F T E D T U M O R S

17. TRAYKOVICH,.D., 0.NGGEz-MONTIEI.,.D., M. RI VI ~RE ,.D.,* ANDM. GUBRIN, .D.

UMOR CELLS CAN BE FOUND IN THE BLOODT f patients suffering from malignanttumors, but they can be found only with greatdifficulty because of the presence of a vastnumber of large benign cells that are notnormally present. These benign cells are fromhematopoietic and connective tissues and arequite often erroneously diagnosed as cancercells. Some authors have named them atypicalcells,@abnormal cells,l~, 4 and large be-nign cells.7 The last expression is consideredthe most appropriate.During the studies carried out on graftedtumors in hamsters,g large benign cells, iden-tical to those observed in human patients,were found in the circulating blood with theproduction of circulating cancer cells andmetastasis. The morphology of such large be-nign cells in hamsters with different tumors isdescribed in this paper.

M AT E R IAL SND METHODSHerbeuval et al.s3 modification of Trayko-vich et al.s technique9 was used to determineleukocyte concentration. The series consistedof 62 venous blood samples from 17 hamsterswith melanoma, 11with fusicellular sarcoma,8 with endotheliosarcoma, and 26 control ani-mals. The tumors were maintained by graft-ing. The blood samples were drawn by rightheart puncture and were diluted in a phos-phate-buffered solution with polyvinylpyrroli-done, sodium citrate, and 2% Formalin. Asaponin solution containing 50% ethyl alco-hol and 2% Formalin was then added bydroplets until the erythrocytes were com-pletely hemolyzed. The mixture was centri-fuged at 2,500 rpm, and the sediment waswashed with Herbeuval and Herbeuvals solu-

From the Instituto Venezolano de Investigaciones* Present address: Institut de Recherches sur le Can-R eceived fo r publication J une 19, 1964.

Cientificas, Apartado 1827, Caracas, V enezuela.cer, Villejuif, France.

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

Of the animals with cancer, 64% showedlarge circulating nonmalignant cells, classifiedas originating from hematopoietic and con-nective tissues. The majority belonged to themegakaryocytic series and some were of themyeloid line. No large cells of the erythro-poietic line were noted. On the other hand,50% of the control animals presented circu-lating nonmalignant cells that were only ma-ture megakaryocytes and endothelial cells.Among the hamsters with melanoma, 8showed metastasis: 5 in the lung, 1 in theliver, and 2 in the lung and liver. Six of the8 had immature cells; 5 of the9 animals with-out metastasis also had immature cells. Fourof the 11 animals with fusicellular sarcomahad metastasis in the lymphatic gland andshowed circulating immature cells; 4 of the7 animals without metastasis also had imma-ture cells. In the8 hamsters with endothelio-sarcoma, metastasis was seen in 6; 3 of the 6had immature forms in the blood, and 1of the2 without metastasis also showed these im-mature forms.Megukuryocytic L i ne Cells. Normal (Fig. 1)and aberrant (Fig. 2) cells were found, as wellas some mature cells (Fig. 5). The sizes variedfrom 50 to 100p. The normal megakaryocyteswere clearly identified because of their largersize. The aberrant cells showed irregularlyshaped nuclei of wide variability and cyto-plasmic tinctorial properties of the normalmegakaryocytes (Fig. 2). The aberrant formscould be considered as originating by meansof extramedullary hematopoiesis. Megakaryo-cytes were also seen in normal animals, but

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No. 12 LARGE ENI GN ELL SN BLOOD F EXPERIMENTALN I M A L S Truykovich et ul. 1611always in mature forms; they were easily iden-tified because of the characteristic plateletformation (Fig. 3).Myeloid L ine Cells. A myeloid reaction wasobserved in the blood of the hamsters withcancer, the lymphocytes having been replacedby polynuclear cells. The differential analysisof the circulating blood of normal hamstersshowed 50 to 96% lymphocytes and 3 to 43%neutrophils. In these animals the differential

count was inverted. Moreover, immature ele-ments, ranging from myeloblasts to metamye-locytes, were noticed; they showed the usualcharacteristics and always exceeded 15 p insize. Figure 4 shows a myelocyte, and Fig. 5shows a metamyelocyte.Mononuclear Cells. Mononuclear cells wereobserved. They showed azurophilic granula-tion in the cytoplasm. The nuclei were moreor 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 . )F IG .2. An aberrant megakaryocyte with very irregular and segmented nuclei from an animal with cancer.F IG .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.)F IG .5. A metamyelocyte showing an unsegmented kidney-shaped nucleus displaced to the periphery withFIG.6. A large mononuclear cell with azurophilic granulation. (May-Grunwald-Giemsa. ~950.)FIG.7. Bare endothelial-cell nuclei. (May-Grunwald-Giems. ~1,5500.)F IG .8. A typical mitosis in anaphase observed in control and cancer animals. (May-Griinwald-Giems.XI500.)F IG .9. An atypical mitosis showing a roselike arrangement. (May-Grunwald-Giemsa. X1,150.)

(May-Grunwald-Giemsa. x950.)

granulation in the cytoplasm. (May-Grunwald-Giemsa. ~1,600.)

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1612 CA NCE R ecember 1964 1701. 17cyte. The mononuclear cells of animals withcancer sometimes appeared larger (18 to 25 p)than normal (Fig. 6) .Endothelial Cells. Endothelial cells wererarely found as single cells but were quiteoften seen in groups. Bare nuclei were morefrequently observed than intact cells. Thecytoplasm was stained pale blue and hadround or elongated nuclei that showed gran-ular chromatin. These cells belonged to largeblood vessels and capillaries (Fig.7).Mitotic F orms. Both typical (Fig. 8) andatypical (Fig. 9) mitotic forms were frequentlyobserved. Romsdahl et al.5 reported the pres-ence of mitotic cells in the blood of micewith experimental tumors. Cells in atypicalmitosis showed irregular chromatin and poly-ploid forms, probably belonging to malignantcells. I n control animals, cells in mitosis werescarce.Undiagnosed Cells. Some cells could not berecognized, either because they had beendamaged or because they were in a state ofdegeneration.

DISCUSSIONOn the basis of the data presented, it hasbeen proved that large benign cells found inthe blood of hamsters with malignant tumorsare similar to those found in human patients.These cells presented a problem in the de-tection of tumor cells in the blood samples oflaboratory animals grafted with tumors. Onthe basis of a relatively wide field of work,Sandberg et al.* concluded that, besides tumorcells, the blood of cancer patients containslarge benign cells, difficult to identify. Bouvier

et al.,1 Herbeuval and Herbeuval,2 and

Herbeuval et al.3 also mentioned difficulty inidentifying cancer cells in the blood of can-cer patients, giving the same reason. Scheininand Koivuniemi7 found these benign cells inthe blood of 82y0of patients suffering fromcancer or certain noncancerous diseases anddescribed the morphology of these cells.The significance of hematopoietic and tis-sue cells in the blood of tumor bearers isworth discussing. Extramedullary hemato-poiesis in those with malignant tumors maybe due to various causes, such as tumor infec-tions, metabolic products, or toxins emanat-ing from the malignant tissue, and metastasisin the bone marrow. I n addition, the myeloidreaction also occurs in the bone marrow ofthose with septicemia.Large benign cells are also seen in the bloodof those with different types of anemia thatare accompanied by extramedullary hemato-poie~is.~herefore, the hematopoietic reac-tion is easy to understand since the bonemarrow is regarded as reacting to many ab-normal conditions of the body as well as tomalignant tumors.

SUMMARYLarge, uncommon benign cells were foundto be present in the blood of hamsters graftedwith metastasis-producing tumors. These hem-atopoietic and nonhematopoietic cells werefound in 67% of the blood samples from lab-oratory animals with malignant tumors. Suchcells were the cause of difficulties and errorsin the diagnosis of malignant cells. These be-nign cells in the blood of experimentalanimals are similar to those found in human

patients with cancer and other diseases.REFERENCES

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HEILLE, ., and GUERCI,0.: Etude du passage descellules hematopoiktiques dans le sang circulant aucours des cancers; importance de la m6gacaryocytPmie.Nouvelle rev. franG. hemat. 2: 619-621, 1962.4. HERBEUVAL,.; HERBEUVAL,., and DUHEILLE,.:Le passage, dans le sang circulant, des amas hbmato-poiktiques, ilots Prythroblastiques, ilots megacaryo-blastiques, ilots mixtes. Nouvelle rev. frunG. hemat. 2:5. ROMSDAHL,. D.; CHU,E. W.; HUME,R., and

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SMITH,R. R.: Time of metastasis and release of circu-lating tumor cells as determined in experimental sys-tem. Cancer 14: 883-888, 1961.6. SANDBERC,. A.; MOORE,G. E., and SCHUBARG,J.. R.: Atypical cells in blood of cancer patients-differentiation from tumor cells. J . N ut. Cancer I nst.7. SCHEININ, . T., and KOIVUNIEMI,. P.: Largebenign cells in circulating blood and their significance

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