VIRUS-INDUCED MURINE LYMPHOMARESEMBLING BURKITT'S TUMOR

JAMES J. BUTLER, M.D., ANTAL SZAKACS, M.D., AND JOSEPH G. SINKOVICS, M.D.

From the Departments of Pathology and Medicine,The University of Texas M. D. Anderson Hospital and Tumor Institute,

Houston, Tex.

In 1958, Burkitt described a tumor observed in Africa that appearedto exhibit characteristic clinical and morphologic findings. Initially, thetumor, which was proved to be a lymphoma,2 was believed to be in-digenous to a specific region of Africa.3 Subsequently, the same mor-phologic changes were observed in tumors found in many other parts ofthe world,"7 though these tumors were less often associated with thedistinctive clinical features described by Burkitt. The belief that Bur-kitt's lymphoma was limited to a fairly well defined climatic and geo-graphic region led investigators to postulate that an arthropod-bornevirus was responsible for its development. As a consequence, differentviruses have been identified in tissues from patients with this lym-phoma.89 Recently, spontaneous tumors having the same morphologicappearance have been reported in dogs 7 and cats10 outside Africa.

In this institution, a lymphoma histologically similar to Burkitt'slymphoma has been induced in Swiss mice by the inoculation of theRauscher virus. A review of the medical literature has failed to disclosea report of a similar observation.

MATERIAL AND METHODSIn August r964, the Rauscher mouse leukemia virus, in the form of cell-free extract

of leukemic mouse spleen, was inoculated into tissue cultures of normal cells fromthe thymus gland and spleen of suckling Swiss mice. Since that time, this culturehas undergone I07 cell passages, by a standard trypsinization procedure. This pro-cedure, as well as the leukemogenic activity of the virus, has been described in detailelsewhere." A portion of the original culture which was not infected with Rauschervirus has also been serially passaged.12

During bioassays of the tissue-culture fluid for leukemogenic activity, a smallnumber of mice developed a lymphoma composed of primitive cells. From one mousewith this lymphoma a cell passage line was initiated. This tumor has been passed inTimco Swiss mice by intraperitoneal and subcutaneous routes; the intraperitonealroute of injection has subsequently been discontinued because of the developmentof hepatitis. The mice given injections of the cell passage line are the subject of thepresent study. The mice were observed for varying periods of time up to io months.

Supported in part by Grant DE 02232 and Contract PH 43-65-628 from the NationalInstitutes of Health, U. S. Public Health Service.

Accepted for publication July 3, 1967.

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Necropsy studies of all the animals were carried out, and print preparations orhistologic sections were made. For histologic examination, tissue was fixed in formalinand sections were stained with hematoxylin and eosin.

For electron microscopic studies, tumor was taken from 3 different passages, at7, io, and I4 days after inoculation of the mice. The fragments were fixed in gluta-raldehyde, postfixed in osmium tetroxide and embedded in Epon. All grids werestained with uranyl acetate and lead citrate and examined with the Hitachi iiBmicroscope.The Swiss mice used in this study were obtained from the Texas Inbred Mouse

Company (Timco), Houston, Tex. The incidence of spontaneous lymphoma in thesemice at I year of age is far less than o.5%.When the term "leukemia" is used herein to describe a tumor in mice, it is utilized

as a general term to denote a neoplasm of the reticuloendothelial system. It does notimply an increase of cells in the peripheral blood.

OBSERVATIONSThe subsequent distribution of the tumors in mice given intraperito-

neal and subcutaneous injections were similar, although masses devel-oped at the site of injection in only the mice which were inoculatedsubcutaneously. Since sections were not obtained in every case and grossdescriptions were limited to only thymus, spleen, lymph nodes, and liver,it was impossible, for this preliminary report, to determine the in-cidence of lymphoma in the mice and the incidence of tumor in the organsof those mice developing tumor.

Histologic evidence of lymph node involvement was present in everycase with lymphoma in which lymph nodes were studied microscopically,but was usually not evident grossly. Where lymph nodes were enlarged,there was rarely more than mild to moderate enlargement. In contrast,only a minority of the mice exhibited thymic involvement, but this wasalways evidenced by thymic enlargement, usually mild in degree. Multi-ple foci of lymphoma were apparent grossly or microscopically in theliver and kidneys of the majority of the animals. Lymphoma of thespleen, usually exhibited by gross enlargement without nodules, waspresent in less than half the mice from which sections of the spleen wereavailable. Four mice had lymphomas in the small intestine or stomach,one in the testes, and one in an ovary. No gross evidence of tumor wasfound in the facial bones.The histologic appearances of the tumors at all sites were similar.

All contained sheets of closely packed, and often cohesive, cells (Fig.i) with large nuclei and a thin, poorly defined rim of cytoplasm. Thenuclei of these cells contained finely distributed chromatin and a smallnucleolus (Fig. 2). Scattered at fairly regular intervals throughoutthese sheets of cells were functioning histiocytes having a large amountof pale cytoplasm (Fig. i). The cytoplasm of the histiocytes containedchromatin fragments, other cellular debris and, rarely, erythrocytes

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(Fig. 2). No histiocytes could be seen in some of the small foci of tumorin the livers and kidneys of the mice, although they were present in thetumor at other sites of involvement. Because of the sheets of cells con-taining histiocytes, the tumor presented the characteristic "starry sky"appearance described in Burkitt's lymphoma. These features of thetumors in the mice were similar to those in a biopsy of Burkitt's lym-phoma of the mandible from a child in this hospital (Fig. 3) and ofreactive centers of hyperplastic lymph nodes (Fig. 4).On electron microscopy, virus particles were present in all 3 passages

of the tumor which were studied; these particles were chiefly extracellu-lar (Fig. 5-7), being more numerous in the younger growths. Severalparticles were of the budding A type, although the majority were of themature C type. Intermediate membrane was clearly apparent in severalparticles of the A type. A more extensive electron microscopic study ofthe tumors is now being conducted.

DIscusSIONThe reactive centers of hyperplastic lymph nodes in both man and

animals resemble Burkitt's lymphoma. This is of particular interest,since an immunologic factor in patients with Burkitt's lymphoma hasbeen postulated,'3 and the reactive centers are believed to play a part inantibody production.'4

In view of all the work done on murine lymphomas and leukemias,spontaneous and induced, it is surprising that the lymphoma describedherein has previously been reported only by Nishizuka and Nakakuki,'5and possibly also by Joske et al.'6 No description or illustration of asimilar tumor appears in Dunn's article on murine lymphoma,'7 or onpathologic descriptions of transmissible tumors, including those pro-duced by the Rauscher 18 and the Friend '9 viruses. Apparently, the tumordescribed by Nishizuka and Kakakuki'5 as having been induced byradiation in C5 7 black mice and a spontaneous tumor in AKR mice wereidentical to the one described herein. Even in their article, the resem-blance of the histologic findings to those of Burkitt's lymphoma was notmentioned. From the photomicrographs alone, evaluation of the tumorreported by Joske et al.-6 is difficult. It is interesting that reovirus 3 hasbeen suggested as the causative agent of this lymphoma.16The causative agent of the lymphoma described herein is not clear.

It is possible that the tumor was initiated by an unrecognized leukemiavirus other than the Rauscher virus, previously proved to be present inthe material injected into the mice,"1 or by a combination of the two.This is particularly true, since, as reported elsewhere,'2 the parent cellline, which has been maintained by serial cell passages but has not been

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intentionally infected with any virus, contains a small number of virusparticles closely resembling the typical murine leukemia virus. Thepresence of another virus or viruses would explain why detailed histo-logic studies of lymphoma produced by the Rauscher virus, reported byDunn and Green,18 have not included this variant of lymphoma.The ultrastructural appearance of virus particles found in our tissue

cultures infected with Rauscher virus 20 was identical with the electronmicroscopic appearance of virus particles in the in-vivo tumor observedin our study. In both, Type C mouse leukemia virus particles predomi-nated. In addition, electron microscopy12 revealed a small number ofvery similar virus particles in the serially passaged parent cell line whichwas not intentionally infected with virus. It is of interest that theRauscher virus-carrying tissue culture of Wright and Lasfargues alsocontained predominantly Type C particles.21

These observations serve to substantiate the view that a distinctionmust be made between the clinical and histologic findings in Burkitt'slymphoma. Although the clinical findings appear to have been associatedwith only a relatively few tumors reported in regions other than Africa,this histologic type of lymphoma is not rare. The histologic featureswhich have been reported as similar to those of Burkitt's lymphoma havebeen found in tumors of dogs 7 and cats,'0 and in tumors of human beingsin all parts of the world.2'47 In the tumors of mice reported herein, thehistologic findings were characteristic of Burkitt's lymphoma, althoughthe distribution of lesions, notably the absence of involvement of jawbones and retroperitoneum were unlike those described from Africa.

SUMMARY

Stem-cell or undifferentiated lymphoma histologically similar to thatpresent in Burkitt's lymphoma in human beings has been observed inSwiss mice subsequent to the injection of a cell passage line started froma single mouse who developed lymphoma following injection of cell freefluid from a tissue culture of the Rauscher virus. Virus particles weredemonstrated in the in-vivo tumor by electron microscopy.

REFERENCES

I. BURKITT, D. A sarcoma involving jaws in African children. Brit J Surg 46:2I8-223, I958.

2. O'CONOR, G. T. Malignant lymphoma in African children. II. A pathologicalentity. Cancer 14:270-283, I960.

3. BURKITT, D. "A Lymphoma Syndrome Dependent on Environment. Part II.Epidemiological Features." In Symposium on the Lympizoreticular Tumoursin Africa, Paris, I963, Roulet, F. C., Ed. International Union Against CancerMonograph Series No. 3. S. Karger, Basel, I964, PP. II9-I36.

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4. O'CoNoR, G. T., RAPPAPORT, H., and SMITH, E. B. Childhood lymphoma re-sembling "Burkitt tumor" in the United States. Cancer z8:4TI-4I7, I965.

5. RYAN, B., CAMPBELL, P. E., and FARAGO, C. Malignant diseases in Melanesianchildren. Med J Aust 1:436-440, I964.

6. BELTRAN, G. Childhood lymphoma in Colombia, South America. Cancer Ig:II24-II30, I966.

7. BRs, G., MURRAY, S. M., and McDONNOUGH, L. T. Sporadic occurrence inJamaica of neoplasms resembling Burkitt's tumour. Lancet 2:6I9-620, I965.

8. BELL, T. M., MASSIE, A., Ross, M. G. R., and WILLIAMS, M. C. Isolation ofa reovirus from a case of Burkitt's lymphoma. Brit Med J Z:I2I2-I2I3, I964.

9. STEWART, S. E., LOVELACE, E., WHANG, J. J., and NGU, V. A. Burkitt tumor:tissue culture, cytogenetic and virus studies. J Nat Cancer Inst 34:3I9-327,I965.

io. SQUIRE, R. A. Feline lymphoma. A comparison with the Burkitt tumor ofchildren. Cancer z9:447-453, I966.

Ii. SINKOVICS, J. G., BERTIN, B. A., and HowE, C. D. Occurrence of low leu-kemogenic but immunizing mouse leukemia virus in tissue culture. Nat CancerInst. Monograph No. 22:349-367. I966.

I2. SINKOVICS, J. G., SZAKACS, A., BUTLER, J. J., HIRANO, M., SHULLENBERGER, C.C., and HoWE, C. D. Pathological conditions associated with the inocula-tion of a long term tissue culture line of a murine leukemia virus. East AfricanMed J. In press.

I3. BURKITT, D. African lymphoma: Observations on response to vincristine sul-phate therapy. Cancer 19:II3I-II37, I966.

I4. FITCH, F. W., and WISSLER, R. W. "The Histology of Antibody Production."In Immunological Diseases, Samter, M., Ed. Little, Boston, I965, pp. 65-86.

I5. NISHIZUKA, Y., and NAKAKUKI, K. A study of gross pathology and histo-pathology of mouse lymphoma. Acta Haemnat Jap 27:456-474, I964.

I6. JOSKE, R. A., LEAK, P. J., PAPADIMITRIOU, J. M., STANLEY, N. F., and WALTERS,M. N. I. Murine infection with reovirus: IV. Late chronic disease and theinduction of lymphoma after reovirus type 3 infection. Brit J Exp Path 47:337-346, I966.

I 7. DUNN, T. B. Normal and pathologic anatomy of the reticular tissue in labora-tory mice, with a classification and discussion of neoplasms. J Nat CancerInst 14:I28I-I433, 1954.

I8. DUNN, T. B., and GREEN, A. W. Morphology of BALB/c mice inoculated withRauscher virus. J Nat Cancer Inst 36:987-IOOI, I966.

I9. METCALF, D., FURTH, J., and BUFFET, R. F. Pathogenesis of mouse leukemiacaused by Friend virus. Cancer Res 19:52-58, I959.

20. GYORKEY, F. Personal communication.2I. WRIGHT, B. S., and LASFARGUES, J. C. Long-term propagation of the Rauscher

murine leukemia virus in tissue culture. J Nat Cancer Inst 35:319-327, I965.

[ Illustrations follow ]

BUTLER, SZAKACS, AND SINKOVICS

LEGENDS FOR FIGURESPhotomicrographs (Fig. I-4) are of sections stained with hematoxylin and eosin.

Electron micrographs (Fig. 5-7) are of sections stained with uranyl acetate and leadcitrate.

FIG. I. Stem-cell (undifferentiated) lymphoma resembling Burkitt's tumor in themouse. X IOO.

FIG. 2. Stem-cell (undifferentiated) lymphoma resembling Burkitt's tumor in themouse (higher power of Fig. i). X 250.

FIG. 3. Stem-cell (undifferentiated) lymphoma, Burkitt's tumor, in a child. Biopsyfrom tumor in mandible. X 250.

FIG. 4. Portion of reactive center in lymph node exhibiting reactive follicular hy-perplasia. X 250.

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FIG. S. Between 3 cells (A, B, and C) are many mature virus particles, mainly Type C. Arrowpoints to particle with double nucleoid. X 79,000. Scale represents I250 A.

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T.A.I.-lim,W .14T.4",." A, 1...,

II'llr ." ..

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6

7

FIG. 6. Virus particle is budding from plasma membrane (arrow) . 140,000. Scale,720 A..

FIG. 6. Virus particle is budding from plasma membrane (arrow) X I40,000. Scale, 720 A.FIG. 7. Virus particle is budding from plasma membrane (arrow) X I75,000. Scale, 570 A.

637Oct. r967