Ultrastructure of malignant melanomas of the choroid

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1 Ultrastructure of malignant melanomas of the choroid Michael J. Hogan and Lynette Feeney Eleven vialignant melanomas of the uveal tract have been studied by light and electron microscopy. Two types of cells were found in the epithelioid tumors: one, a darker type, contained large amounts of free RNP particles in single granules or in clusters, many smooth membranes and vesicles, and numerous mitochondria; the lighter type had similar cytoplasmic components, but in a lesser amount, especially in the number of free RNP particles. The epithelioid cells were closely packed, in contrast with the findings of light microscopy, and the cell walls were smooth and straight. In mixed tumors it was more difficult to distinguish spindle and epithelioid cells, except by prior identification with stained 1 n sections. The three spindle cell B tumors showed a very uniform pattern in low-power field with the electron microscope, but in higher magnifications exhibited considerable variety in cytoplasmic composition, with numerous large mitochondria, many smooth-surfaced vesicles, and an amorphous component in the cytoplasm. In contrast with the epithelioid cell there is more rough-surfaced endoplasmic reticulum, and an absence of free RNP particles in the cytoplasm. All tumors contained a variable number of macrophages, characterized by large masses of cytoplasmic pigment. It was not possible to determine whether these were true reticuloendothelial cells, or tumor cells which had phagocytized pigment. The origin of the melanin granules was also not revealed by this study. Possible modes of origin of these tumors were considered. None of the cells resembled Schwann cells of normal human beings, nor did they exactly resemble normal melanocytes. In addition to pigmentation, many similiarities to the normal melanocyte could be detected in the spindle B type cells. ^allender 4 classified malignant melanomas of the uveal tract according to their cytologic and morphologic features. This classification was later 5 ' 24 found to be useful in determining the prognosis in affected patients. The following types were described: spindle A, spindle B, mixed, From the Department of Ophthalmology and the Francis I. Proctor Foundation for Research in Ophthalmology, University of California Medical Center, San Francisco, Calif. This investigation was supported by Research Grants Nos. B-1229 and B-1782 from the Institute of Neurological Disease and Blindness, National Institutes of Health, United States Public Health Service, Bethesda, Md. epithelioid, fascicular, and necrotic. The epithelioid and necrotic tumors are the most malignant and the spindle types the least so. Francois and associates 11 ' 12 described the electron microscopic features of two melanomas of the choroid. Both were of the spindle B variety; one was pigmented, the other was not. Both tumors had essentially the same ultrastructure. The pigmented tumor had pigment granules which were not homogeneous. Each granule was composed of finer particles which were embedded in a less dense material. Some of these granules were surrounded by a delicate, smooth membrane. An additional 544

2 Volume 1 Number 4 Ultrastructure of malignant melanomas of choroid 545 feature was the presence in the cytoplasm of larger rounded (about 0.5 /x) or oval spaces surrounded by a delicate membrane and containing melanin particles in a less opaque ground substance. The findings suggested that the rapid development of the malignant cells caused abnormal formation of pigment granules, and that the vacuolar spaces in the cytoplasm were incompletely formed pigment granules. Wellings and Siegel 21 studied 10 biopsy specimens of melanomas from human skin and described melanin granule formation in the Golgi apparatus. The smallest melanin granules were found in the Golgi zone, arising by deposition of a dense material within the small vesicles of the Golgi apparatus. The authors 23 later described the morphology of the same tumor cells. In general, the cells corresponded to previous descriptions of electron microscopic studies of other normal and neoplastic cells. The melanin granules had an average diameter of 300 m/x, were circular or oval, uniform in density, and occasionally showed a substructure of finer granules or rods. Macrophages were found in the tumors and showed conspicuous differences from the melanocytes, having smaller nuclei and nucleoli, fewer mitochondria, less ergastoplasm, less Golgi material, and in the presence of very large ovoid or circular pigmented masses. The internal granular and rodlike structure of the phagocytized granules was more prominent and more easily seen than in those of the tumor cells. The rod-shaped internal units of phagocytized melanin granules sometimes showed a longitudinal beaded appearance or periodicity which was not apparent in the melanin granules of the tumor cell. Seiji, Fitzpatrick, and Birbeck 17 studied melanoma pigment granules of the mouse and pigment epithelium of the chick and correlated the electron microscopic appearance with enzymatic content. They concluded that the melanin granules and mitochondria are distinct cytoplasmic constituents of melanin-forming cells. They proposed the term "melanosome" for the characteristic enzymatically active particle which is the site of melanin formation. The melanosome is a cell organelle with an internal structure which ranges in size from 0.1 to 0.4 fx. The melanin granule is 0.4 to 20 fi in diameter and is composed of the melanoprotein polymer. Melanosome will be used to describe the small striated or granular melanin particles seen in the tumors. During the past 2 years we have collected 11 malignant melanomas of the uveal tract for study. All specimens were obtained from adult human eyes. Comparisons will be made of the cytology as seen with the light microscope in paraffinembedded sections of 10 to 12 fx, in methacrylate-embedded sections of 1 n, and in thin sections with the electron microscope. Materials Seven of the melanomas involved the choroid, 2 the choroid and ciliary body, 1 the ciliary body alone, and 1 the iris. Of the 7 choroidal melanomas 6 were of the mixed cell type, and one was of the spindle cell B variety. One of the 2 tumors involving both the choroid and ciliary body was of the mixed type, and the other was of the spindle cell B variety. The ciliary body tumor was an epithelioid tumor, and that of the iris was a spindle cell B type. One tumor of the ciliary body was thought at first to be a spindle cell A type, but on electron microscopy all nuclei were found to contain nucleoli. Restudy of thinner sections showed the tumor to be a spindle cell B type. Methods At the time of operation all eyes were opened quickly and in a direction determined by the position of the tumor. A portion of the tumor was removed for formalin fixation and embedding in paraffin, and additional pieces were placed in cold (4 C.) veronal buffered (ph 7.4) osmic acid (OsO 4 ). After 3 to 5 minutes' fixation they were divided into 1 to 2 mm. portions and transferred to fresh fixative for 2 to 3 hours. They were then dehydrated and embedded in methacrylate, and sectioned with a Porter-Blum microtome or an LKB ultratome. The sections were placed on carbon-coated copper grids, and stained with lead hydroxide. All were viewed with the RCA EMU-3 electron microscope. The paraffinembedded portion of the tumor was sectioned and stained with hematoxylin and eosin. Other

3 546 Hogan and Feeney Investigative Ophthalmology August 1962 sections were bleached with potassium permanganate (KMnO<) before staining. Comparisons were made betwen the hematoxylin and eosin paraffin sections, bleached paraflbn sections, 1 /i thick methacrylate sections stained with the Goodpasture-MacCallum stain, and the 1/40 p electron microscope sections. Results The paraffin sections were used to determine the type of melanoma and the degree of pigmentation. Six of the choroidal melanomas were of the mixed type and one was of the spindle B variety. Five tumors were moderately pigmented (2 to 3 plus) and 2 were lightly pigmented (1 plus). One of the tumors which involved the choroid and ciliary body was a very lightly pigmented spindle cell B tumor; the other a moderately pigmented (3 plus) mixed cell tumor. The one ciliary body tumor was a lightly pigmented (1 plus) epithelioid tumor. The iris tumor was a lightly pigmented (1 plus) spindle cell B tumor. The identification of cell types was less easy in the 1 p thick stained methacrylate sections. Spindle cells in the spindle cell B tumors could be identified because of the uniformity of the cells and their heavy brandling processes (Fig. 1). In the mixed tumors it was less easy to differentiate the spindle cell B type from the epithelioid because the Iaclc of cohesion of epithelioid cells was less evident in osmium-fixed tissues, and the spindle cells showed fewer and smaller branching processes (Fig. 2). The epithelioid cells in the 1 n methacrylate sections could be recognized by their size, large nuclei, nucleoli, and abundant cytoplasm (Fig. 3). Macrophages were present in all tumors, but were especially numerous in the heavily pigmented tumors. Their large masses of phagocytized cytoplasmic pigment and other material often obscured the nucleus. Bleached paraflbn sections showed these cells to have most features of the adjacent tumor cells. Many sections showed that the lumina of blood vessels contained macrophages. & Fig. 1. (EM 140 B) Spindle cell B melanoma of the iris. One micron thick methacrylate section. A group of spindle B type cells from the central portion of the tumor. (Goodpasture-MacCallum stain. x630.) Fig. 2. (EM 77 Ti) Mixed cell melanoma of the choroid and ciliary body. One micron thick methacrylate section. Epithelioid cells near the right margin and spindle cells centrally. Large macrophage to the right of center. (Goodpasture- MacCallum stain. x630.) Fig. 3. (EM 134 T 2 ) Epithelioid cell melanoma of ciliary body. One micron thick methacrylate section. (Goodpasture-MacCallum stain. x630.)

4 Volume 1 Number 4 UItrastructure of malignant melanomas of choroid 547 N D ' t- JS8&W Figs. 4 and 5. For legends see page 551.

5 r 548 Hogan and Feeney Investigative Ophthalmology August 1962 R M \ M Fig. 6. For legend see page 551. '/*

6 Volume 1 Number 4 Ultrastructure of malignant melanomas of choroicl 549 N Figs. 7 and 8. For legends see page 551.

7 550 Hogan and Feeneij Investigative Ophthalmology Augtist 1962 Figs. 9 and 10. For legends see page 551.

8 Volume 1 Number 4 Ultrastructure of malignant melanomas of choroid 551 Fig. 4. (EM 77-66) Electron micrograph of an epithelioid cell in a mixed cell melanoma of the choroid and ciliary body. The nucleus contains a large ropelike nucleolus. The density of the nuclear membrane is caused by an accumulation of chromatin along the inner nuclear membrane. The cytoplasm contains numerous small mitochondria and some large empty saccules of endoplasmic reticulum. G, Golgi apparatus. ME, Melanin granules. (xl0,000.) Fig. 5. (EM ) Low-power electron micrograph of an epithelioid cell melanoma of the ciliary body showing the "dark" (D) and "light" (L) cells. A layer of collagen (C) separates groups of cells into bundles. (x4,600.) Fig. 6. (EM 65-94) Electron micrograph of a portion of an epithelioid cell. A portion of the nucleus (N) is seen at upper left. The cytoplasm contains several mitochondria (M), melanosomes showing several stages in melanization (MS), clusters of RNP particles or ribosomes (R), smooth-surfaced vesicles (V), and a portion of the Golgi apparatus (G) consisting of smooth-surfaced lamellae and vesicles. (x35,000.) Inset shows melanosomes at higher magnification. 1 and 2, Unmelanized melanosomes showing fibrillar matrix. 3 and 4, Longitudinal section showing beaded appearance after some melanin is deposited on the fibrils. 5, Cross section of a melanosome. (x65,000.) Fig. 7. (EM ) Electron micrograph of a portion of a dark epithelioid cell containing many melanosomes (MS), free RNP granules (R), and vesicles (V) in an amorphous cytoplasmic matrix. Intercellular fibrils (F) measuring about 100 A in diameter appear at upper right. The nucleus (N) occupies the lower portion of the picture. M, Mitochondria. (x26,000.) Fig. 8. (EM 140-2) Electron micrograph of portions of three cells from a spindle B melanoma. The cytoplasm contains many mitochondria (M) and some organized endoplasmic reticulum (ER). The Golgi apparatus (G) is visible in the cell on the left. Large "empty" saccules (S) of endoplasmic reticulum are seen in two of the cells. N, Nucleus. (x20,000.) Fig. 9. (EM65-84) Electron micrograph showing the variety in the cytoplasmic contents of spindle B melanoma cells. The cell on the left contains rough-surfaced endoplasmic reticulum (ER) in the form of vesicles and short lamellae and two melanosomes (MS) are visible. The cell on the right contains long parallel lamellae of rough-surfaced endoplasmic reticulum and no melanin. On the extreme right mature melanin granules (MG) are seen in the cytoplasm of a third spindle B cell. N, Nucleus. M, mitochondria. (x23,000.) Fig. 10. (EM 65-87) Electron micrograph of a cell which is found in all melanomas and which has the characteristic features of a macrophage. Melanin granules (MG) and melanosomes (MS) are found within large membrane-bound sacs in the cytoplasm. In addition, the cell contains the usual cytoplasmic organelles: M, Mitochondria. ER, Endoplasmic reticulum. G, Golgi apparatus. N, Nucleus. Collagen fibers (C) lie in the matrix outside the cell. (x9,000.) Epithelioid types of cell. Unlike the epithelioid cells seen in paraffin sections, those seen in the electron microscope were close together (150 A intercellular space). The cell boundaries were straight. The nuclei were very large in proportion to the surrounding cytoplasm. The chromatin was evenly dispersed except for a denser layer along the inner nuclear membrane (Fig. 4). The nucleoli were large and dense and were composed of a tangled mass of bands or ropes which had a uniform width of 0.1 to 0.15 p.. On the basis of the appearance of the cytoplasm, both dark and light cells were seen in various fields (Fig. 5). No intermediate forms were observed. The difference was produced by the quantity of cytoplasmic components. The dark cells were loaded with ribonucleoprotein (RNP) particles which occurred singly or in clusters and rarely were attached to membranes (Fig. 6). Smooth membranes and vesicles were abundant even in areas distant from the Golgi apparatus. A free amorphous component occupied much of the cytoplasm, and numerous mitochondria were seen (Fig. 7). The light cells exhibited the same cytoplasmic components but to a lesser degree, particularly in the number of free RNP particles. This tumor contained intercellular fibrils

9 552 Hogan and Feeney Investigative Ophthalmology August 1962 with 600 to 640 A periodicity typical of both collagen and reticulin, and bundles of fine (100 to 180 A) unbanded fibrils. Elastic fibers also were seen in some areas. The melanin content and morphology in the epithelioid tumors varied. Nearly every cell contained melanosomes. Lighter pigmented cells showed a wider spectrum of pigment forms: melanosomes, densely striated granules, and larger solid, homogeneous melanin granules. Spindle B cells. The cells in these tumors showed the same close alignment and straight borders as the epithelioid cells, except at the very periphery of the tumor or where poor fixation was evident. The nuclei were more oval and many showed extensive convolutions of the nuclear membrane. In proportion to the amount of cytoplasm, they were smaller than those of the epithelioid cells. There was no chromatin layer at the inner nuclear membrane, as was seen in the epithelioid cells. The cytoplasm of these cells did not show the quantitative differences which were seen in the epithelioid cells, i.e., in a low-power field all cells looked alike. Aside from this, however, there was generally more variety in cytoplasmic composition among the spindle B than among the epithelioid cells (Fig. 8). Like the epithelioid, the spindle B cells had numerous mitochondria and many smooth-surfaced vesicles. There also was a similar amorphous component in the cytoplasm. In contrast with the epithelioid cell, there was more rough-surfaced endoplasmic reticulum, i.e., the RNP particles were attached to the cytoplasmic membranes. At the same time tliere was not the striking abundance of free RNP particles in the cytoplasm. The rough endoplasmic reticulum was in the form of vesicles and very short double membranes; extensive parallel arrays were not seen (see mixed tumors). Most cells showed large saccules of rough endoplasmic reticulum the contents of which appeared to have been dissolved by fixation procedures or sublimed by the electron beam. There were frequent bizarre forms of endoplasmic reticulum, usually in the form of spirals. The Golgi vesicles of one tumor (139) contained a dense amorphous material like that seen in secretory cells. The melanin content was quite variable. One tumor (139) contained no pigment except for an occasional branching melanocyte with mature granules. Another (140) showed only an occasional cell with finely striated melanosomes. Most of the cells of another (814) contained abundant large (about 0.5 ^u.) pigment granules of the finely striated type. All those of still another (838) contained numerous solid or densely striated melanin granules. Collagen rarely was seen in these tumors except around the blood sinuses. Mixed tumors. The 7 mixed tumors were composed of epithelioid and spindle B cells in varying proportions. It was frequently difficult, however, to identify a cell as epithelioid or spindle B because more cytoplasmic variation was observed in these tumors. Cells with the following characteristics of epithelioid cells as observed in 1 ^ sections by light microscopy were selected for study: large nuclei, large fibrous nucleoli, extensive cytoplasm, and lack of alignment in bundles. Electron micrographs of these cells showed the features of dark epithelioid cells, with vesicles of all descriptions as the predominant feature. The vesicles were of all sizes, surrounded by thick and thin membranes, and appeared empty or contained amorphous material, particles, and tiny vesicles. They might or might not be studded with RNP particles, and some definitely contained melanin. Large saccules of endoplasmic reticulum which were apparently empty were a common feature, similar to those of spindle cells. Nearly every cell contained large lipid droplets. Mitochondrial sizes varied, but a given cell had uniform-sized mitochondria. Cells which most nearly fitted the morphologic concept of spindle cells seen by light microscopy also showed much more cytoplasmic variation than those of the

10 Volume 1 Number 4 Ultrastructure of malignant melanomas of choroid 553 pure spindle B tumors. These cells could be placed in 2, and possibly 3, classes: (1) those with the rough endoplasmic reticulum beautifully arranged in long parallel lamellae, and (2) those with a few short lamellae of endoplasmic reticulum and many free RNP granules occurring singly and in rosettes (Fig. 9). A possible third category contained fingerlike projections of endoplasmic reticulum around the mitochondria. The various cells suggested a progression of dedifferentiation, possibly toward the epithelioid type. In addition, all cells contained lipid droplets and large empty saccules of endoplasmic reticulum. The melanin content varied from tumor to tumor and from cell to cell. Two tumors described as moderately to heavily pigmented in histologic sections (65 and 77) showed many cells with but few melanin granules, others with numerous melanosomes, too small to be resolved by the light microscope, and still others which contained many large solid granules. In one tumor (133), only a rare cell contained pigment and it was of the mature or solid type. All the mixed tumors contained some connective tissues, but in varying amounts. Typical collagen, reticulin, and fine fibrils were often seen. One tumor (65) also contained elastic fibers. One of these tumors (133) had groups of cells bound into fasicles 20 to 30 p. wide by strips of connective tissue 1 to 2 yu. wide. The cells had interdigitating borders, unlike the 3 spindle B tumors. Macrophages. In all the tumors, regardless of cell type, a cell occurred which had a very characteristic and interesting morphology (Fig. 10). The cell was very large (usually larger than the epithelioid cell) and frequently showed branching. The cytoplasm contained pleomorphic, rounded bodies composed of all forms of melanin, from microgranules to large solid granules. These bodies resembled the inclusions of macrophages seen in other tissues, and particularly those in pigmented tissues. The inclusions were readily visible by light microscopy, some being as large as a red blood cell. In addition to the forms of melanin, the inclusions contained a particulate material which was less dense than the melanin microgranules. This material was evenly dispersed throughout the inclusion and obscured its limiting membrane. The cytoplasm of the macrophage differed from that of the epithelioid cells in that it had large mitochondria and few free RNP particles. It would be difficult to distinguish this cell from the spindle B cells on the basis of cytoplasmic content. Blood sinuses. The walls of the blood sinuses were less than 0.2 p. thick and resembled capillaries in structure. They were composed of a thin layer of endothelium, thin basement membrane, and a few collagen fibers. Macrophages were frequently found in the lumen just inside the wall. In one tumor two macrophages were observed entering the sinus through gaps in the endothelial layer. Discussion The ultrastructure of normally pigmented cells has occupied the attention of many investigators for the past several years. The melanocytes of the skin,-> 3 > s > 10> is, 2G hajj-i retina, 25 13> 20 and uveal tract"' of human beings have been studied. The melanocyte of the skin of various animals also has been studied by Selby. ls Comparison of the normal melanocyte with the malignant melanoma cell shows some differences. The nuclei are similar in structure, and, except for the greater size of nucleus in relation to cytoplasm in the epithelioid cell, cannot be distinguished. The nucleolus of the epithelioid cell is outstanding in its size and ropelike pattern. The cell membranes are similar except that in the melanocyte they are more irregular. The membranes of the spindle cell and epithelioid cell also are similar. Some differences are noted between the cytoplasm of the normal and of the malignant cells. The epithelioid cell contains very numerous RNP granules, mostly lying free, in

11 554 Hogan and Feeney Investigative Ophthalmology August 1962 contrast with the melanocyte and spindle cells, which have RNP in considerable amounts but attached to the membranes of the endoplasmic reticulum. The epithelioid cell also contains large amounts of smooth membranes and vesicles, even in areas remote from the Golgi apparatus. The melanin granules in the cytoplasm of all the malignant cells vaiy in amount, size, and composition. Some cells show large granules of uniform density, size, and distribution, similar to those of normal melanocytes. Others show very fine granules of a size beyond the resolution of the light microscope. Still others show small granules which vary in size and distribution, some with a solid structure, or with fine granules or striated particles. The variegated granules more nearly resemble those seen in "immature" melanocytes of the human choroid 9 and those of the developing mouse. 15 The findings in these specimens show the spindle B cells to resemble more closely those described by Wellings and Siegel 23 in skin melanomas. They found considerable endoplasmic reticulum with attached RNP particles. In contrast with their findings, the Golgi apparatus in our specimens did not appear more prominent than in the normal melanocyte. The smooth vesicles and sacs in the cytoplasm of most malignant cells, however, might be a part of the apparatus, for we could not establish the origin of the melanosomes since they were frequently found in the periphery of the cell at some distance from the Golgi zone. This study and a previous one 9 suggest that the melanin granule arises in vesicles in the cytoplasm as described by Mover 15 in embryonic mice. An initial formation of fine parallel filaments occurs in the vesicle, followed by alignment of osmiophilic melanin granules, like beads on a string, along these filaments. Cross sections of such vesicles show a fine granulation, and longitudinal sections show them to be oval and to contain the fine filaments with their melanin granules. Eventually, the whole vesicle becomes filled with melanin. In this respect Wellings and Siegel also found the granules in melanoma cells to be composed of granular and rod-shaped structures. It is less easy to compare the spindle B cells of our tumors with the two described by Francois and associates. 11 ' 12 The cloudy swelling of the mitochondria in their second case suggests degenerative changes caused by other disease, or poor fixation. Otherwise, the endoplasmic reticulum with its attached RNA granules and other organelles were similar to the spindle B cells of our tumors. The cytoplasm of some cells in the first case described 11 also showed the curving elements of endoplasmic reticulum which were found in some spindle B cells of our mixed tumors. In addition to their "normal" pigment granules, they observed cytoplasmic spaces which were bound by membranes and contained melanin granules and a less opaque granular material. These spaces are considerably larger and the melanin microgranules are more dispersed than any seen by us or by other investigators. The macrophages in these tumors, initially described by Dalton and Felix, 6 may be a true reticuloendothelial cell which has gathered pigment liberated from adjacent melanoma cells, or a neoplastic cell which behaves as a phagocyte. The light microscope study of unbleached and bleached sections shows they are indistinguishable from melanoma cells. The electron microscope shows them to be large and to contain pigment and other material in very large masses in the cytoplasm. It is not possible to determine whether these cells are melanoma or reticuloendothelial cells. We have observed the same type of cell as a normal component of the human choroid, 9 which suggests that it is more likely a macrophage than a melanoma cell. The interstitial tissues and blood spaces of all these tumors were found to be much as described with the light microscope. No nerves were encountered in any of the tumors.

12 Volume 1 Number 4 Ultrastructure of malignant melanomas of choroid 555 We wish to thank Miss Miriam Duschkin for her valuable technical assistance. REFERENCES 1. Birbeck, M. S. C, Mercer, E. H., and Barnicot, N. A.: The structure and formation of pigment granules in human hair, Exper. Cell Res. 10: 505, Charles, A., and Ingram, J. T.: Electron microscope observations of the melanocyte of the human epidermis, J. Biophys. & Biochem. Cytol. 6: 41, Clark, W. H., and Hibbs, R.: Electron microscopic studies of the human epidermis. The clear cell of Masson, J. Biophys. & Biochem. Cytol. 4: 679, Callender, C. R.: Malignant melanoma tumors of the eye: A study of histologic types in 111 cases, Tr. Am. Acad. Ophth. 36: 130, Callender, C. R., Wilder, H. C, and Ash, J. E.: Five hundred malignant melanomas of the choroid and ciliary body followed five years or longer, Am. J. Ophth. 25: 962, Dalton, A. J., and Felix, M. D.: Phase contrast and electron micrography of the Cloudman S91 mouse melanoma, in Gordon, Myron, editor: Pigment cell growth, New York, 1953, Academic Press, Inc., pp Dalton, A. J., and Felix, M. D.: The electron microscopy of normal and malignant cells, Ann. New York Acad. Sc. 63: 1117, Drochmans, P.: Electron microscope studies of epidermal melanocytes, and the fine structure of melanin granules, J. Biophys. & Biochem. Cytol. 8: 165, Feeney, L., and Hogan, M. J.: Electron microscopy of the human choroid. I. Cells and supporting structures, Am. J. Ophth. 51: 1057, Fitzpatrick, T. B., Brunet, P., and Kukita, A.: The nature of hair pigment, in Montagna, William, and Ellis, Richard A., editors: The biology of hair growth, New York, 1958, Academic Press, Inc. 11. Francois, J., Rabaey, M., and Lagasse, A.: Examen au microscope electronique d'un sarcome de l'uvee, Ophthalmologica 137: 65, Francois, J., Rabaey, M., and Lagasse, A.: Electron-microscopic data on pigment formation in melanoblastomata, A. M. A. Arch. Ophth. 64: 377, Holmberg, A.: Ultrastructure of the ciliary epithelium, A. M. A. Arch. Ophth. 62: 935, Karrer, H. E.: The ultrastructure of mouse lung: The alveolar macrophage, J. Biophys. & Biochem. Cytol. 4: 693, Moyer, H.: Electron microscope observations on the origin, development and genetic variation of melanin granules in the eye, in Smelser, George K., editor: The structure of the eye, New York, 1961, Academic Press, Inc. 16. Reese, A. B.: The differential diagnosis of malignant melanoma of the choroid, A. M. A. Arch. Ophth. 58: 477, Seiji, M., Fitzpatrick, T. B., and Birbeck, M. S. C: The melanosome: A distinctive subcellular particle of mammalian melanocytes and the site of melanogenesis, J. Invest. Dermat. 36: 252, Selby, C. C.: An electron microscope study of the epidermis of mammalian skin in thin sections. I. Dermo-epidermal junction and basal layer, J. Biophys. & Biochem. Cytol. 1: 429, Dvorak-Theobald, G.: Neurogenic origin of choroidal sarcoma, Arch. Ophth. 18: 971, Tousimis, A. J., and Fine, B. S.: Ultrastructure of the iris: An electron microscopic study, Am. J. Ophth. 48: 397, Wellings, S. R., and Siegel, B. V.: Role of Golgi apparatus in the formation of melanin granules in human malignant melanoma, J. Ultrastruct. Res. 3: 147, Wellings, S. R., Barishak, R., and Siegel, B. V.: Cytological studies of human malignant melanoma cultured in vitro, Cancer Res. 20: 347, Wellings, S. R., and Siegel, B. V.: Electron microscopy of human malignant melanoma, J. Nat. Cancer last. 24: 437, Wilder, H. C., and Paul, E. V.: Malignant melanoma of the choroid and ciliary body: A study of 2,535 cases, Mil. Surgeon 109: 370, Yamada E., Toduyasu, K., and Iwaki, S.: The fine structure of retina studied with electron microscope. II. Pigment epithelium and capillary of the choriocapillary layer, J. Electronmicroscopy 6: 42, Zelickson, A. S., and Hartmann, J. F.: An electron microscopic study of human epidermis, J. Invest. Dermat. 36: 65, 1961.