Retinal neoplasia and dysplasia. I. Induction by feline leukemia virus

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1 Retinal neoplasia and dysplasia. I. Induction by feline leukemia virus D. M. Albert, M. Lahav, E. D. Colby, J. A. Shadduck, and D. N. Sang Feline leukemia virus, a naturally occurring C-type virus (oncorna virus), was injected systemically and intraocularly into fetal and newborn kittens. A light microscopy study of the resultant ocular tumor changes was carried out. One animal developed an intraocular tumor of apparent retinal origin. Eight animals showed intraocular neoplastic cell infiltration. The most common ocular change observed was retinal dysplasia. Sequential histologic examination of enucleated eyes showed diffuse intraocular inflammation after viral inoculation. In the developing retinas progressive disorganization and necrosis were documented, idth subsequent reorganization into cell clumps and dysplastic rosette structures. The retinalpigment epithelium showed proliferation and intraretinal migration. The mature retina exhibited full-thickness folds and tubes associated with infoldings of the retinal pigment epithelium. Key words: feline leukemia virus, C-type virus, retinal neoplasia, retinal dysplasia. iral infection of immature tissues may result in a spectrum of responses. These include acute necrotizing inflammation; at times, a slow insidious inflammatory process; and in certain instances a persisting From the Howe Laboratory, Massachusetts Eye and Ear Infirmary, Boston (Drs. Albert and Sang), Department of Ophthalmology, Hadassah Medical School, Jerusalem (Dr. Lahav), Dartmouth Medical School, Hanover, N. H. (Dr. Colby), and Southwestern Medical School, Dallas (Dr. Shadduck). Supported by Grant EY of the National Eye Institute, National Institutes of Health, Bethesda, Md. Reprint requests: Daniel M. Albert, M.D., Howe Laboratory of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, 243 Charles St., Boston, Mass latent infection. All these responses may damage germinal cells, with subsequent altered embryogenesis, malformation, and atrophy of tissues. 1 In the case of oncogenic viruses, the viral genome may cause malignant transformation of the immature cells with tumor formation. - Retinal dysplasia is a congenital retinal anomaly which may appear spontaneously or be observed after naturally occurring or experimental viral, 3 " 7 chemical, 8 - IJ or physical 10 ' " insults to the developing retina. Retinal dysplasia is characterized by anomalous development of the retina with dysplastic rosettes, retinal folds, and gliosis. A clinical and histopathologic classification has recently been published. 1 - A number of viruses such as bovine mucosal diarrhea disease virus, 3 bluetongue virus, 4 lympho-

2 326 Albert et al. st. Ophthalmol. Visual Sci. April 1977 The importance of viruses as agents which cause neoplastic changes in experimental animals is well established.- DNA viruses can cause malignant transformation of cells in tissue culture 13 ' 14 and tumors of retinal origin in experimental animals. 13 ' 15 The purpose of this report is to describe the occurrence of dysplastic and neoplastic changes of an oncogenic RNA virus, feline leukemia virus, on the immature retinal tissue with respect to dysplastic and neoplastic changes of the retina. Fig. 1A. Intraocular inflammation 5 days after intraocular inoculation with FeLV. Note inflamed and thickened iris (I), cataractous lens (L), and inflammatory cells in vitreous (V). (H & E; xl.8.) Fig. IB. Inflammatory cell infiltration of the cornea (c) and iris (I) 14 days after intrauterine infection. Note anterior synechia, destruction of Descemet's membrane (DM), and necrosis of the iris pigmented epithelium (PE). (H & E; x200.) cytic choriomeningitis virus, 5 adenovirus, 0 and canine herpesvirus 7 have been shown to cause retinal dysplasia in experimental animals. Dysplastic rosettes showing apparent photoreceptor differentiation and bearing a similarity to Flexner-Wintersteiner rosettes of retinoblastoma have been described. 12 Materials and methods Cats. The cats used were from random-bred strains without known spontaneous occurrence of leukemia or malignant lymphoma over a period of 2 to 4 years. The animals were maintained individually in outdoor holding pens and were time-mated 1511 as needed for the experiment. Virus. The virus used was Rickard's strain of feline leukemia virus (FeLV), which was supplied by Drs. C. G. Richard and J. E. Post. The virus had been isolated from a spontaneous case of cat leukemia and underwent 45 passages in cat embryo cells and five complete passages in human embryo cells which were transformed by the viral infection. The viral pool consisted of unconcentrated tissue culture fluid collected from a whole human embryo cell (HWE 2836) infected with FeLV. Mode of infection. Three groups of animals were used in the experiment. In the first group, cats were time-mated, and on day 40 of pregnancy, under general ether anesthesia, the abdominal cavity was surgically opened and the uterus exteriorated. A 0.1 ml. amount of viral pool containing 10~ 4 tissue culture infective dose of Rickard's FeLV was injected intraperitoneally into each fetus. The uterus was replaced, and the abdominal wall sutured. Four fetuses were aborted and studied at 2 days (three fetuses) and 8 days (one fetus) after infection. Eight kittens were sacrificed or died at sequential intervals after delivery at periods up to 57 days after infection. In the second group seventeen kittens were inoculated on the day after birth. With the animal under ether anesthesia, the lid adhesions were separated by gentle traction with forceps. A puncture was made with a 27-gauge needle at the pars plana on the temporal side. The needle was pointed posteriorly into the vitreous cavity under direct observation, with care not to injure the retina or the lens. A 0.1 ml. amount of tissue culture fluid containing 10" 4 tissue culture infective dose of Rickard's FeLV was injected into the right and left eyes of each ani-

3 Volume 16 Dumber 4 Retinal neoplasia and dysplasia. I 327 Fig. 2A. Mixed population of lymphocytes (L) and histiocytic cells (H) with mitotic figures (M) are seen in the iris of an animal 28 days after intrauterine infection. (H & E; x400.) Fig. 2C. Reticulum cells (arrows) within the iris of the kitten with intraocular tumor. TG, Tumor giant cells. (H & E; x400.) Fig. 2B. Intraocular tumor with extrascleral extension 16 days after intraocular infection. (H & E; xl.8.) mal. A cotton-tipped applicator was held in place over the puncture site for 3 minutes following injection. Six kittens died spontaneously at different time intervals after infection, with signs of encephalitis. The rest were sacrificed at times from the sixth to the forty-eighth day after inoculation by intravenous injection of pentobarbital. The third group, serving as a control, consisted on eight noninjected kittens which were sacrificed by intravenous injection of pentobarbital at Fig. 2D. Transitional area between normal retina and area of neoplastic transformation which arises in the inner nuclear layer in the kitten with intraocular tumor. (H & E; x200.) variable time intervals between the fourth and thirtieth day of age. Animals that developed systemic symptoms of encephalitis or seemed moribund during the experiment underwent autopsy. Eyes from all groups

4 328 Albert et al. Invest. Ophthalmol. Visual Sci. April 1977 Fig. 2E. Flexner-Wintersteiner rosettelike structures (R) within the same tumor. Note the similarity to the rosettes of retinoblastoma. (H & E; xl,000.) Table I. Ocular changes in kittens infected in utero No. of animals Days post infection Cornea Iris CB Lens Choroid Vitreous Iritis Cyc- Cata- Choroid- PAS litis ract itis ± ± ± - ± Vitreitis Optic nerve Retina Optic Disorganization neuritis of embryonal cells; inflammation + Disorganization; inflammation; cellular clusters + Early layering; disorganization, inflammation, folds, subretinal exudate, cellular balls; spaces in external layers + Disorganization; inflammation; 3 layer folds; cellular clusters in ou.ter retinal layer with eosinophilic material in center - Atrophy of all retinal layers Pigment epithelium Diffuse destruction Disorganization Disorganization Patchy destruction under disorganized retina Patchy destruction under disorganized retina

5 Volume 16 Number 4 Retinal neoplasia and dysplasia. I 329 Fig. 2F. Dysplastic (nonneoplastic) rosettelike structures in an area of atrophic and disorganized retina, which represent attempt at reorganization (arrows), in the kitten with the intraocular tumor. (H&E;x400.) Fig. 3B. Folds of atrophic retina 23 days after intraocular inoculation. Persistent mild inflammatory cell infiltration within the choroid (CH) and infolding of retinal nonpigmented epithelium (arrows) over the tapetum (T) are seen. (H & E; x400.) Fig. 3A. Early retinal fold with subretinal exudate (arrow) is seen 2 days after intrauterine inoculation. Mild choroidal inflammatory cell infiltration is seen (CH). The immature neural retina shows slight disorganization, with necrosis of the retinal pigment epithelium (thick arrow). (H & E; xloo.) were enucleated immediately after death and fixed in 10 percent phosphate-buffered formalin for 12 hours. The tissue was then examined under the dissecting microscope and processed for histologic examination by standard techniques. Microscopic serial sections (6 jtt) were cut with a microtome and stained with Harris' hematoxylin and eosin (H & E) and periodic acid-schiff (PAS) stains. Selected sections were stained with Brown and Brenn stain for bacteria. All sections were examined under the light microscope, and the degree of inflammation, any malformation, and evidence of tumor formation in the various ocular structures was recorded. Fig. 3C. Marked foldings of the retina 41 days after intraocular inoculation. Note thickening of the iris due to persistent inflammation and a cataractous lens. (H & E; xl.8.) Results Despite the varying route of infection, animals injected with virus peritoneally in utero and the group infected intraoeularly postnatally were observed to have generally similar distribution and severity of ocular inflammation (Tables I and II). Mild to severe evidence of intraocular inflammation is present in all" eyes, includ-

6 330 Albert et al. st. Ophthalmol. Visual Set. April 1977 Fig. 3D. Fixed retinal folds and tubular structures (arrows), which represent folds cut on the bias, are seen 41 days after intraocular inoculation. Note the absence of subretinal exudate and the inflammatory cells in the choroid and retina. (H &E; x200.) Fig. 4B. Neural retina 5 days after intraocular inoculation with FeLV. Mild infiltration with inflammatory cells, cellular disorganization, and acellular spaces in the external nuclear layers (arrow) are seen. (H & E; x200.) Fig. 4A. Minimal cellular disorganization of the outer retinal layers is seen over normal retinal pigment epithelium (PE) 2 days after intrauterine inoculation. (H & E; x400.) ing keratitis, peripheral anterior synechia, chamber angle infiltration, cataracts, iridocyclitis, choroiditis, retinitis, vitreitis, optic neuritis, and meningitis (Figs. 1A and IB). In the fetuses examined, the inflammatory cells appear as large immature leukocytes. In the fully developed kittens the inflammatory infiltrate, consisting of well-differentiated lymphocytes, and plasma cells are seen (Fig. IB). In four animals killed 6, 13, 23, and 48 days after intraocular infection and in three animals killed 18 days after intrauterine infection, an additional cell type is present: these appear histiocytic and show hyperchromatic nuclei Fig. 4C. Marked retinal inflammatory cell infiltration, necrosis, and disorganization with formation of folds, and subretinal exudate which contains pigment-laden macrophages (arrow). Note the choroidal inflammation (CH) and destruction of the overlying pigment epithelium 12 days after intrauterine inoculation. (H & E; xloo.) and eosinophilic cytoplasm. Some pleomorphism of nuclei and occasional mitotic figures can be seen (Fig. 2A). In one animal that was infected postpartum and sacrificed on the sixteenth day after infection, a large tumor occupies the vitreous cavity and invades the sclera and optic nerve. In several areas, the tumor appears to arise from the retina (Fig. 2B). This tumor is composed of pleomorphic cells having hyperchromatic nuclei with clumping of chromatin and large

7 Volume 16 Number 4 Retinal neoplasia and dysplasia. I 331 «^%» jr*:~z?ir- -fawn, y Fig. 4D. Disorganization of the external retinal layers 40 days after intrauterine inoculation. Note rosettelike structures arranged around a central area which is filled with fibrillary material (arrows). Structure which resembles the external limiting membrane of the retina is seen (double arrow). (H & E; x400.) Fig. 5C. Focal infolding of the retinal pigment epithelium (arrow) under a retinal fold. (H & E; x400.) Fig. 5A. Normal neural retina and retinal pigment epithelium of a 13-day-old kitten. (H & E; xloo.) Fig. 5D. Focal proliferation of the retinal pigment epithelium (arrows) under a retinal fold. (H &E; x200.) Fig. 5B. Focal necrosis of the retinal pigment epithelium. Note total absence of cells (one arrow); loss of pigment (two arrows); and cellular hypertrophy (three arrows). (H & E; xl,000.) nucleoli; multinucleated tumor cells are frequently seen. In most cells the cytoplasm is abundant, eosinophilic, and vacuolated (Fig. 2C). In the areas interpreted as showing malignant transformation of the retina (Fig. 2D), structures composed of cells arranged around a central lumen, limited by a structure resembling the external limiting membrane, are seen (Fig. 2 ). In other places the retina is disorganized and destroyed by the infiltrating cells.

8 332 Albert et al Invest. Ophthalmol. Visual Set. April 1977 Table II. Ocular changes in kittens infected postnatally No. of animals Days post infection Cornea Iris CB Lens Choroid Vitreous ,' 16 Extensive invasion by tumor cells; little inflammation Optic nerve Retina Early differentiation; disorganization; inflammation; folds Disorganization of mature retina; inflammation; atypical cells; retinal nuclei in clusters, budding into inner nuclear layer; 3 layer folds; subretinal exudate Mostly destroyed by tumor; cellular clusters seen; 1 and 3 layer folds and tubes; rosettelike structures seen Migration of outer retinal nuclei into inner retinal layers; folds of outer layer; fullthickness folds Disorganization in external retinal layers; clusters of cells," budding into inner nuclear layer; lumen formation with eosinophilic material; atypical histiocytes; few folds Disorganization; clusters of external nuclear cells, budding into inner nuclear layer; eosinophilic material in center; chorioretinal scars with complete atrophy; 3 layer folds and tubes Less disorganization; clusters of cells with eosinophilic material; fused folds of external nuclear layers; 3 layer folds and tubes Pigment epithelium No change Patchy destruction; folded under retinal folds Destroyed by tumor Proliferated under folds; focal destruction; hypertrophy Focal destruction Focal destruction Destroyed in areas; proliferated under folds; intraretinal migration

9 Volume 16 Number 4 Retinal neoplasia and dysplasia. I 333 Gliosis and folding of the retina are seen as well, with apparent reorganization of the outer nuclear layer into rosettelike structures (Fig. 2F). In two of the kittens that were infected in utero, multiple malignant neoplasms composed of lipidcontaining cells were found in visceral organs; no ocular involvement was noted. These tumors were diagnosed as liposarcomas. The retinal inflammation and malformation generally correspond in severity with the degree of intraocular inflammation seen elsewhere in animals receiving intraocular injections neonatally. Four out of 12 animals injected in utero showed marked keratitis, uveitis, and optic neuritis, but comparatively little retinal inflammation or disturbance of retinal architecture. Three distinct forms of retinal malformations are noted in both the animals infected in utero and those infected intraocularly after birth: (1) formation of retinal folds, tubes, and rosettes; (2) intraretinal cellular disorganizations; and (3) atrophy of the retina. Retinal folds are seen by 2 days after infection in association with the accumulation of subretinal exudate (Fig. 3A). These folds may involve the full thickness of the retina or may be seen as partialthickness folds involving only the outer retinal layers. The pigment epithelial layer is enfolded in a few areas where retinal folds are seen (Fig. 3B). Subsequently the subretinal fluid disappears and the folds assume a "fixed" appearance (Fig. 3C). Some of these folds fuse at the base and, in a few planes of sectioning, assume the appearance of a transverse section through a tube surrounded by two or three cellular layers (Fig. 3D), the so-called "two or three layer rosettes." 12 In some animals these retinal folds and tubes are associated with severe disorganization and apparent rearrangement of the cells within the retina, and in others the retinal layers are preserved with minimal disorganization and atrophy. The earliest intraretinal cellular changes are disorganization of the external retinal layers and inflammatory cell infiltration. In the animals which were infected in utero, the initial inflammatory cell infiltration is very mild (Fig. 4A). On the second to fourteenth day after infection, marked disorganization of the cellular layers of the retina, and most particularly of the external nuclear layers, is noted (Fig. 4C). By the twenty-eighth day, clusters of cells with central lumen which contain eosinophilic outer segment-like material can be seen (Fig. 4D). The maturation rate of the retina is unaltered as compared to the control animals. The retinas of the animals that were infected postpartum show inflammatory cell infiltration and slight disorganization of the intraretinal cellular arrangement, with spaces free of cells in the external nuclear layer (Fig. 4B). Massive inflammatory cell infiltration and retinal necrosis are seen beginning on the sixth day after infection. At 2 weeks after infection, reorientation of the cells in the outer retinal layers occurred in areas with the appearance of clsuters of retinal cells. The nuclei of these clusters resemble those of the outer nuclear layer. These changes are similar to but less extensive than those observed in the animals infected in utero. At 3 weeks after infection, clusters of cells with central lumen and centrally located eosinophilic outer segment-like material are seen. In most animals these changes are associated with the formation of retinal folds and tubes and rosettes. Almost no evidence of inflammation or necrosis is evident within the retina in animals sacrificed 3 weeks or more after intraocular viral injection. Atrophy of all retinal layers can be seen as a late sequela, 6 to 8 weeks after infection. Retinal atrophy is present more often in association with retinal folds and rosette formation (Fig. 3B). In early postinfection stages, large areas of pigment epithelium destruction associated with choroidal inflammatory cell infiltration are seen. Vacuolization, depigmentation, and later necrosis of the pig-

10 334 Albert et al. Invest. Ophthalmol Visual Sri. April 1977 ment epithelial cells are present (Figs. 5A and 5B). The retinal pigment epithelium is occasionally folded under retinal folds (Fig. 5C). At 2 weeks after infection, patchy areas of hypertrophy, proliferation, and atrophy of pigment epithelial cells are noted (Fig. 5D). These changes typically are seen under areas of retinal malformation. However, normal retina over abnormal pigment epithelium, and abnormal retina over normal pigment epithelium, can be found. Rarely, intraretinal migration of pigment epithelial cells is present, but these have no clear-cut association with foci of photoreceptor cell clusters. Discussion Spontaneous malignancies of the hematopoietic system in cats is a relatively common occurrence 1 * 1 " 18 and is associated with FeLV. 19 This virus appears to cause a variety of both neoplastic is and nonneoplastic diseases in the infected cats. 20 Intraocular involvement, however, is rarely reported. 17 Resulting neoplasms include, most commonly, alimentary lymphosarcoma, multicentric lymphosarcoma, thymic lymphosarcoma, lymphatic leukemia, 21 ' - fibrosarcoma, 23 " 25 and liposarcoma. 2 " The nonneoplastic diseases associated with FeLV include nonregenerative anemia, immune complex glomerulonephritis, and, importantly, an immunosuppressive syndrome consisting of thymic atrophy and depression of the cell-mediated immune system, predisposing cats to intercurrent infections. 20 ' Fatality rates secondary to immune suppression may be much over 50 percent in experimentally induced FeLV infections. 21 The incidence of naturally occurring infection with FeLV in the general cat population, extrapolated from studies of the incidence of measured antibody to the virus, is 5 to 50 percent, depending on the degree of contact between animals. The incidence of leukemias and lymphosarcomas in the general cat population, presumed to all be associated with FeLV, is approximately 0.05 percent of the cat population. 21 In addition, there is a high percentage of spontaneous abortions in FeLV-positive cats. 28 FeLV, like other viruses of the oncornavirus group, has been shown to contain RNA-dependant DNA polymerase.'"' The C-type particle associated with this virus 30 and its associated neoplasms is 90 to 115 m/x in diameter and is morphologically similar to that found in murine leukemias. 21 ' 31:3 - This particle has been isolated from spontaneous " 30 and experimental cases 34 ' " 3S of feline malignancies and has been serially propagated in cell culture. 81 ' 3!) - 40 Documentation of the presence of the virion has been by electron microscopic, immunologic, 41 ' 42 and tissue culture methods, 32 in addition to the enzymatic studies. Under experimental conditions, FeLV has been shown to be capable of transforming heterologous cells in tissue culture from many species, including dogs, 25 rabbits, 25 primates, 43 " 40 and man The expression of the viral infection as a neoplasm may be the result of one or more of a number of factors. It may reflect a breakdown in the efficiency of the immune surveillance system of the host versus both the virus itself and the tumor cells; the ability of the virus to induce subsequent immune suppression in the host; and the dose of the virus. In this system, high antibody titers to virus-associated cell membrane antigens 4 * 5 have been shown to be associated with failure of tumor induction or regression of established tumors, and there is a significant incidence of spontaneous regression. 4 " The latent period in experimentally induced tumors with FeLV is variable. This period may range from 4 to 7 weeks 20 ' 50 up to 4 years or more, 21 depending on the age of the animal and the dose of virus used/' 1 The earliest histopathologic change that can be detected in the infected tissue is that of histiocytic proliferation. 23 This phenomenon was seen in five of our experimental animals. In addition, plasma cells and lymphocytic infiltration of the uveal tissue were marked in most of our experimental animals during the initial period after infection. Generally, in experimentally

11 Volume 16 Number 4 Retinal neoplasia and dysplasia. I 335 infected animals most of the clinical forms of the disease, 15 " 17 ' 2:) varying from leukemia 1 " to lymphosarcoma, 24 can be produced by the same virus. It appears from the present experiment that retinoblasts are susceptible as well to neoplastic transformation by FeLV. It has been previously shown in vitro 13 ' 14 and in vivo that retinal cells can be.transformed by oncogenic DNA viruses. It may be concluded therefore that both types of viral genome are capable of inducing retinal neoplasia. In most of the experimental intraocular viral infections carried out to date, the viral insults produced anomalous retinal development 3 ' 7 rather than a neoplasia. The changes seen are retinal inflammation, retinal detachment, formation of folds, and gliosis of the folds into tubular structures. In many instances, the retinoblasts alter their normal differentiation and orientation, either secondarily to extensive retinal damage or as a result of interruption of the normal inductive mechanisms controlling the development and orientation of these cells. The retinoblasts that survive the acute infection are observed to form josettelike structures, with the final appearance being that of cells similar to those found in the external nuclear layer arranged around a central lumen. The lumen is usually limited by a structure resembling the external limiting membrane of the neural retina and contains material which resembles photoreceptor outer segments. 12 This structure is similar in appearance to the Flexner-Wintersteiner rosettes which can be found in retinoblastoma and in retinal tumors induced by DNA adenovirus 15 and in the present study by FeLV. A common denominator to all the processes that result in the formation of the so-called "dysplastic rosettes" is the occurrence of extensive cellular damage. In most instances, the derangement of subsequent development and differentiation is limited. Attempts at repair and organization are seen, but the cells do not show an uncontrolled growth. However, as a result of certain infections such as DNA or RNA oncogenic virus infections in the proper host, loss of the control over the normal cellular growth pattern occurs, with subsequent neoplastic proliferation. In the present study, the presence of the two forms of response, i.e., that of reparative reaction, or "retinal dysplasia," and that of uncontrolled cellular proliferation, or neoplastic growth, are not mutually exclusive and can be seen simultaneously in the same eye; the human counterpart has also been seen. 5 -' Hitherto, DNA virus-induced intraocular tumors resembling retinoblastoma have been reported. 13 1S- Sl * The induction of an intraocular tumor by FeLV, however, is of particular importance in that this is an RNA virus-induced tumor and therefore may serve as a more convincing animal model for the study of retinoblastoma, on the basis of the following facts: (1) its resemblance to retinoblastoma histopathologically, with evidence of rosettes morphologically similar to Flexner-Wintersteiner rosettes; (2) the obvious association of this tumor with a known RNA virus which has been shown to contain RNAdependent DNA polymerase (retinoblastoma has also been shown to contain an enzyme with similar template specificity, 51 both in vivo and in vitro); and (3) the significant incidence of spontaneous regression in both the FeLV-associated spontaneously occurring tumors and spontaneously occurring retinoblastoma. Extraocular FeLV-induced neoplasia has been extensively studied as an appropriate model of host-tumor interaction and the immune response to neoplasia : 5r '" 5T viral proteins and antigens have been isolated, and experimental vaccines ss have been developed. Intraocular FeLV-induced tumors provide us with an equally promising model for a similar study. REFERENCES 1. Johnson, R. T.: Effect of viral infections on the developing nervous system, N. Engl. J. Med. 287: 599, Albert, D. M., and Rabson, A. S.: The role of viruses in the pathogenesis of ocular tumors, Int. Ophthalmol. Clin. 12: 195, 1972.

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