Extraocular Orbital Tumors Induced by Human Adenovirus Type 12 ...

Extraocular orbital tumors induced byhuman adenovirus type 12 in hamsters

Noritsugu Mukai and Shoji Kobayashi

Intravitreous inoculation of human adenovirus type 12 (Huie strain) in 59 newborn hamstersproduced 12 extraocular orbital tumors 27 to 106 days after a single injection. Histopathologicstudies suggest that these tumors are an undifferentiated malignant neoplasm that closelyresembles a neuroepithelioma that can be induced in the central nervous system by the samevirus. Although the neoplastic cells had different architectural patterns, the basic structureshowed a uniform perivascular festooning, accompanied by the formation of pseudorosettes.Serial sectioning indicated that the ciliary ganglia and parasympathetic efferent fibers weremissing in all 12 eyes. Hypertropic nerve fibers embedded in tumor mass were occasionallyseen. Electron microscopic observations suggest that these tumors are poorly differentiatedmalignant counterparts of an epithelial tumor with its nidus in neural primordia.

Key words: human adenovirus type 12, intraocular inoculation, neuronal primordiain retrobulbar adnexa, undifferentiated neurogenic tumor.

Since Trentin, Yabe, and Taylor1 dis-covered that human adenovirus type 12induced a high incidence of malignanttumors in newborn hamsters, this uniqueoncogenic DNA virus has been used bya number of scientists to produce neo-

From the Wesley C. Bowers Laboratory of Phar-macology and Experimental Pathology, Depart-ment of Retina Research, Retina Foundation,Boston, Mass. 02114.

This work was supported by the MassachusettsLions Eye Research Fund, Inc., by PublicHealth Service Research Grant EY-00227 of theNational Eye Institute, by Fight for Sight Inc.,and by Research to Prevent Blindness, Inc.

Manuscript submitted Aug. 17, 1972; manuscriptaccepted Oct. 17, 1972.

Reprint requests to Retina Foundation OphthalmicLibrary, 100 Charles River Plaza, Boston, Mass.02114.

plasms.2"7-19 This communication is the firstreport of virus-induced tumors arising fromorbital soft tissues. The nidus of thesetumors has not been determined, but theirmorphology suggests that they should bedesignated as an undifferentiated neuro-genic neoplasm derived from the primordiain the retrobulbar space.

Materials and methodsPreparation of concentrated virus fluid. Human

adenovirus type 12 (Huie strain, Flow Labora-tories, Baltimore, Md.) was cultured in HeLa cells.Monolayer-cultures were prepared in 250 ml. plasticflasks (Falcon) with Eagle's basal medium supple-mented by 15 per cent calf serum. Cultured mono-layers of HeLa cells were infected with adeno-virus fluid and incubated for 48 hours with Eagle'sbasal medium supplemented by 2 per cent calfserum. After the medium was decanted, thoseHeLa cells that yielded the adenovirus-specificcytopathic effect were carefully collected with apoliceman (No. 8433). After three cycles of freez-

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186 Mukai and Kobayashi Investigative OphthalmologyMarch 1973

Fig. 1. Grayish-red, firm tumor mass (arrow) ex-panding laterally to the temporal edge of the orbit;77 davs after virus inoculation.

ing and thawing, the collected HeLa cells werecentrifuged at 2,500 r.p.m. for 10 minutes in orderto eliminate cell debris. Virus-containing fluid wasstored at - 20° C.

Inoculation of virus fluid. Pregnant hamsters(LVG:LAK strain) were supplied by the Lake-view Hamster Colony, N, J. A single intravitreousinoculation of 0.01 ml. virus fluid, 103r> to 104-5

TCID"'" HeLa cells per 0.1 ml. was made in theleft eyes of 39 hamsters within 24 hours afterbirth. The injection was made under an operatingmicroscope with a fine hypodermic needle (70M,Metropolitan Supply, Cambridge, Mass.) connectedto a microsyringe (No. 710-N, Hamilton).

Twelve control animals were inoculated withsupernatant fluid from nonvirus-infected HeLacells cultured in the same medium as that used forthe preparation of virus fluid. All animals werefed a balanced diet of pellets (General Biochemi-cals).

hnmunofiuorescence microscopy. Adenovirus-specific T antigen(s) was examined by the directimmunofluorescein microscopic procedure3 withcryosections of tumors and primary culture cells.

Light microscopy. Formalin-fixed paraffin sec-tions and frozen cryostat sections of tumor tissuewere prepared. For cytologic identification, hema-toxylin and eosin, iron-hematin, phosphotungsticacid hematoxylin, and Holmes' silver stain wereused. Cryostat sections were examined histochemi-cally for NADH and NADPH tetrazolium oxido-reductases, ATPase, alkaline and acid phosphatases,luxol fast blue, and lithium carbonate silver.

Electron microscopy. Samples were taken froma solid, non-necrotic tumor and immersed in 2.5per cent glutaraldehyde. All specimens were post-fixed in 1.0 per cent osmium tetroxide in PCXbuffer pH 7.2 for 2 hours. After dehydration, eachwas embedded in Epon-812. Ultra-thin sectionscut with a LKB-Ultrotome were stained withnranyl acetate and lead citrate. Electron micro-

grams were made with the Philips 200 and 300electron microscopes.

Results

Twelve of 59 hamsters inoculated withadenovims developed extraocular orbitaltumors 27 to 106 days after virus injection.Macroscopically, all 12 tumors, which oc-cupied the temporal part of the orbits,appeared almost identical, showing a gray-ish-white smooth surface (Fig. 1, arrow).On sectioning, the tumor mass tended toshow necrosis and to hemorrhage. Theoptic nerve and sclera showed no neo-plastic encroachment. Solid tumors alsodeveloped in the liver of 3 animals. Notumors developed in control animals ob-served for 250 days.

Immunofluorescence microscopy. Withfluorescein microscopy tumor cell cyto-plasm responded characteristically to theadenovirus-specific antibody from the se-rum of hamsters with adenovirus-inducedtumors.

Light microscopy. The tumors were onthe temporal side of the eye. There was notumor cell infiltration in the posterior seg-ment of the eyeball or the optic nerve.Serial sectioning indicated that the ciliaryganglia, parasympathetic efferent fibers,and long ciliary nerves were missing in all12 eyes. In some cases, enormously hyper-trophied nerve fibers and lemmoblasticcells were found within the tumor.

The major part of the tumor was denselypacked with small hyperchromatic cellsthat often formed a uniform perivascularcell-wreath (Fig. 2, white arrow). A peri-vascular festooning pattern was occasional-ly seen around larger blood vessels. Rapid-ly degenerating tumor cells, interspersedwith well-preserved cells around bloodvessels gave the tumor sections a trabecu-lar or papillary appearance. Tumor cellswere mixed with plasma cells and poly-morphonuclear leukocytes (Fig. % blackshort arrow) in the retrobulbar soft tissues.High power views of cells that possessedonly a scanty cytoplasm consistently showedthat the basic cytoarchitectural feature


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Extraocular orbital tumors 187

Fig. 2. Large mass of retrobulbar tumor showing characteristic cell pattern. Numerous smallblood vessels with nucleus-free cuffs give tumor its peculiar leopard skin appearance (whitearrow). Marked plasma cell infiltration and leukocytes {black arrow) appear in remaining partof retrobulbar tissue. There is no neoplastic encroachment in optic nerve and retina. (Hema-toxylin and eosin, x40).



Fig. 3. Perivascular festooning and numerous pseudorosettes suggest tumor may be neurogenicin origin. (Phosphotungstic acid hematoxylin, x250).

was perivascular festooning closely asso-ciated with pseudorosettes (Fig. 3). Mitoticfigures were very common, and giant cellswere found occasionally. Collagen or retic-ulin formation was meager. Silver stainsoutlined round or elongated ovoid nucleiand nucleoli; debris in the center of pseudo-rosettes stained intensely with silver (Fig.4).

Histochemical findings. Only the scantyrim of tumor cell cytoplasm responded toNADH and NADPH tetrazolium oxido-reductases. ATPase was very active in therim of cytoplasm and in the vascular walls;alkaline phosphatase was positive only invascular channels. Acid phosphatase wassporadically positive in some degeneratingtumor cells. Luxol fast blue stain wasmoderately positive in the cytoplasm.Lithium carbonate silver did not showdifferentiated neurofibrillar elements.

Thin sections (2 to 3/J,) from epon-em-bedded material, stained with toluidineblue, showed the cytologic characteristicsof solid tumors. Round or ovoid nuclei usu-ally had several basophilic nucleoli. The

nuclear membrane occasionally appearedindented. Tumor cells fused, forming acytoplasmic syncytium. Intracytoplasmic,basophilic, round inclusions were ratherfrequent, and mitotic figures were alsocommon.

Electron microscopy. The tumor cellsvaried in size and shape and showed aslightly elongated, ovoid, or cone-shapedcell body with an ovoid nucleus that oc-cupied a large portion of the cell. Eachcell appeared to be connected to cytoplas-mic processes (Fig. 5). Cytoplasmic or-ganelles were scarce; they showed a fewmitochondria and small vesicles. Free ribo-somes were frequent, but granular endo-plasmic reticulum was rare. Poorly de-veloped Golgi's lamellae and vesicles wereobserved only occasionally. Irregularlyshaped lipid droplets and osmiophilicheterogeneous granules were frequent.Bizarre inclusion bodies (Fig. 6, arrows),consisting of multiple vesicle-like particles3

were occasionally found within the cyto-plasm as well as in the interstices of thetumor cell syncytium.


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Extraocular orbital tumors 189

Fig. 4. Characteristic argent affinity of tumor cell nuclei. Mitotic and degrading cells, as wellas debris in the center of pseudorosettes show intense argent affinity. (Lithium carbonatesilver, x250.)

Discussion

The results described are, we believe,the first report of neoplastic transformationof the retrobulbar space caused by adeno-virus type 12. The remarkably uniformhistopathologic characteristics of thesetumors justify their designation as a coun-terpart of human embryonic neuroepithe-liomas for which cytologic criteria are wellestablished.0"11 An example of poorly dif-ferentiated neuroepithelioma invading theorbit is demonstrated by Hogan and Zim-merman.11 The formation of perivascularcell-wreaths and pseudorosettes7'12'19 ischaracteristic (Figs. 2,3, and 4). Cytological-ly} the poorly differentiated tumor cells re-semble an undifferentiated human neuro-blastoma.12'ti! Furthermore, ultrastructurallesions such as osmiophilic heterogeneouscorpuscles and inclusion bodies are sugges-tive of tumors derived from differentiatingneural precursor cells of neural crest ori-gin.14 The inclusion bodies described in thispaper are frequently encountered in nor-

mally developing neuronal precursor cellsin the retina of perinatal rodent embryos.

Although there is no direct evidence forasserting a neuroectodermal origin of thistumor, it is significant that the ciliaryganglia were absent in all 12 intraorbitaltumors. Occasional appearance of hyper-trophic nerve fibers within the neoplasticmass may also imply its neurogenic origin.Indirectly, this suggests that the nidus ofthe tumors is in neural precursor cellsmigrating toward the normal location ofthe ciliary ganglia at the time of inocula-tion. It seems reasonable to assume thatthe neuronal precursor populations thatare migrating along the ciliary nerve fromthe brain would be particularly susceptibleto adenovirus type 12.

Green and co-workers15 suggested thatthe incorporation and transcription of atleast part of the viral genome are obliga-tory steps in adenovirus oncogenesis.Strohl, Rabson, and Rouse10 have alsostressed that the importance of target cell



Fig. 5. Electron microscopic picture of undifferentiated tumor cells showing a few cytoplasmicorganelles. Elongated cells possess cytoplasmic processes forming syncytial pattern. (x7,000.)

determinants in the morphology of tumorsproduced by DNA viruses may be ordainedby their viral genome. However, it is un-certain whether or not the phenotype ofthe remarkably uniform adenovirus tumormorphology is determined by its viralgenome, no matter how different cells invarious tissues are involved as a target inadenovirus tumorigenesis. It is puzzlingthat Strohl, Rabson, and Rouse producedhighly uniform adenovirus-typical tumorsby using a clonal line of the fibroblasticBHK 21 cells as a target for virus infec-tion. If we insist on a monistic theory toexplain the oncogenesis, the tumor theyobtained from a purely clonal line offibroblastic cells should be designated as asarcoma. However, tissue culture explants

of neural retinas tested by Albert, Rabson,and Dalton17 produced tumors closely re-sembling human retinoblastomas withoutrosettes following abortive adenovirus in-fection. Only a pluralistic theory of adeno-virus oncogenesis could explain that thesensory retinas used for tissue culturemight be transformed into a sarcoma afteran abortive adenovirus infection.

In the present study, three tumors de-veloped in the liver, probably as a resultof insinuation of the intraocularly injectedvirus fluid into the bloodstream. It hasbeen previously documented that intra-venous inoculation of adenovirus 12 innewborn hamsters results in tumors in theliver of 100 per cent of the cases.is Themorphology of these tumors was almost


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Extraocukw orbital tumors 191

Fig. 6. Electron-opaque heterogeneous particles in the apical cytoplasmic processes are fre-quent. Unusual inclusion bodies composed of vesicles (arrows) are also common. Note evenlydistributed free ribosomes in the cytoplasm, (xl.1,000.)

identical to that encountered in the retro-bulbar tumors. There is no evidence thatthese hepatic tumors are neurogenic inorigin. Nevertheless, ubiquitously distrib-uted neural precursor cells in developingnewborn animals may be selectively sus-ceptible to this unique DNA virus, as hasbeen demonstrated in the central nervoussystem.3- •• " • in

The authors wish to express their appreciationto Professor H. M. Zimmerman, Chief, Depart-ment of Pathology, Montefiore Hospital MedicalCenter, New York, and Dr. L. E. Zimmerman,Chief, Ophthalmic Pathology Branch, ArmedForces Institute of Pathology, Washington, D. C,for valuable advice and criticism. Serial sectionswere made by Miss Ruiko Kato.

REFERENCES1. Trentin, J. J., Yabe, Y., and Taylor, G.: The

quest for human cancer viruses, Science 137:835, 1962.

2. Huebner, R. J., Rowe, W. P., and Lane, W.T.: Oncogenic effects in hamsters of humanadenovirus types 12 and 18, Proc. Nat. Acad.Sci, 48: 2051, 1962.

3. Ogawa, K., Tsutsumi, A., Iwata, K., et al.:Histogenesis of malignant neoplasm inducedby adenovirus type 12, Gann 57: 43, 1966.

4. Yabe, Y., Ogawa, K.; Awata, K., et al.: Effectof injection of adenovirus type 12 in adulthamsters, Acta Med. Okayama 20: 147, 1966.

5. Spjut, H. J., Van Hossier, G. L., and Trentin,J. J.: Neoplasms in hamsters induced byadenovirus type 12, Arch Pathol. S3: 199,1967.

6. Neiders, M. E., Weiss, L., and Yohn, D. S.:



A morphologic comparison of tumors pro-duced by type 12 adenovirus and by theHA-12-1T line of adeno-12 tumor cells, Can-cer Res. 28: 577, 1968.

7. Mukai, N., and Kobayashi, S.: Primary brainand spinal cord tumors induced by humanadenovirus type-12, J. Neuropath Exper.Neurol. (Submitted for publication.)

8. Pope, J. H., and Rowe, W. P.: Immuno-fluorescent studies of adenovirus 12 tumorsand of cells transformed or infected by adeno-viruses, J. Exp. Med. 120: 577, 1964.

9. Wolter, J. R., and James, B. R.: Adult typeof medulloepithelioma of the ciliary body, Am.J. Ophthalmol. 46: 19, 1958.

10. DeBuen, S., and Gonzalez-Angulo, A.: Dik-tyoma (embryonal medullo-epithelioma). Re-view of the literature and report of a case,Am. J. Ophthalmol. 49: 606, 1970.

11. Hogan, M. J., and Zimmerman, L. E., editors:Tumors of the uveal tract, in OphthalmicPathology; an Atlas and Textbook. Ed. 2.Philadelphia, 1962, W. B. Saunders Co., pp.438-441. (Figure 371, 7).

12. Zimmerman, H. M.: Brain tumors: Theirincidence and classification in man and theirexperimental production, Ann. N. Y. Acad.Sci. 159: 337 (Fig. 13), 1969.

13. Misugi, K., Misugi, N., and Newton, W. A.:Fine structural study of neuroblastoma,

ganglioneuroblastoma, and pheochromocytoma,Arch. Pathol. 86: 160, 1968.

14. Mukai, N., and Ishida, Y.: Tumors of thenervous system, In Sugano, H., and Kobay-ashi, H., editors: Pathology of Tumors,Tokyo, 1971, Asakura Co., pp. 824-870. (InJapanese.)

15. Green, M., Fujinaga, K., Pina, M., et al.:Molecular basis of viral oncogenesis, In Ex-ploitable Molecular Mechanisms and Neo-plasia, Baltimore, 1969, Williams and WilkinsCo., pp. 479-506.

16. Strohl, W. A., Rabson, A. S., and Rouse, H.:Adenovirus tumorigenesis: Role of the viralgenome in determining tumor morphology,Science 156: 1631, 1967.

17. Albert, D. M., Rabson, A. S., and Dalton, A.J.: In vitro neoplastic transformation of uvealand retinal tissue by oncogenic DNA viruses,INVEST. OPHTHALMOL. 7: 357, 1968.

18. Yohn, D. S., Weiss, L., and Neiders, M. E.:A comparison of the distribution of tumorsproduced by intravenous injection of type 12adenovirus and adeno-12-tumor cells, CancerRes. 28: 571, 1968.

19. Mukai, N., and Kobayashi, S.: Undifferentiatedintraperitoneal tumors induced by humanadenovirus type 12 in hamsters, Am. J. Pathol.69: 239, 1972.


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