Investigative Ophthalmology & Visual Science, Vol. 32, No. 9, August 1991 Copyright © Association for Research in Vision and Ophthalmology

Immunohistochemical Properties of Human

Evidence of Type 1 Origin

Paul E. Cutarelli,* Uros R. Roessmann,* Robert H. Miller, f Charles S. Spechr4 and Hans E. Grossniklaus§

The peroxidase-antiperoxidase method was used to study ten surgically obtained human optic nerve (pilocytic ). All tissues were formalin fixed and paraffin embedded. Primary antisera included glial fibrillary acidic protein (GFAP), HNK-1 (type 1 astrocyte precursor marker), A2B5 (type 2 astrocyte precursor marker), S-100, vimentin, myelin basic protein (MBP), laminin, keratin, cytokeratin, epithelial membrane antigen (EMA), and -specific enolase (NSE). Neo- plastic in optic nerve gliomas stained with GFAP, HNK-1, S-100, and vimentin. Oligoden- drocytes and myelin sheaths stained for MBP, and NSE stained surviving axons in the tumors. Neo- plastic astrocytes did not stain for A2B5, keratin, cytokeratin, EMA, or laminin. These results suggest that human optic nerve gliomas (pilocytic astrocytomas) arise from type 1 astrocytes. Invest Ophthal- mol Vis Sci 32:2521-2524,1991

As a result of the variable histopathologic appear- with the identification in the rat central nervous sys- ance of optic nerve glioma, several classifications of tem of a macroglial progenitor cell capable of forming the origin of the proliferating astrocyte comprising astrocytes and .4-5 To characterize these tumors have arisen.1"3 The light microscopic ap- more fully the cellular elements present in optic nerve pearance of certain cells in optic nerve glioma encour- glioma and to identify the origin of the proliferating aged some authors to interpret these tumors as oligo- astrocyte, we immunostained ten human optic nerve dendrogliomas.1 The detection of neurofibrils in these gliomas. cells by immunohistochemical methods, the findings of astrocytic features by electron microscopy, and the Materials and Methods histologic similarity between optic nerve gliomas and cerebellar astrocytomas by light microscopy, taken Ten surgically obtained human optic nerve gliomas together, provided the basis for classifying these tu- were studied. All specimens were fixed in 10% forma- mors as pilocytic astrocytomas (juvenile type).1"3 lin and embedded in paraffin. The diagnosis of pilocy- Some authors, nevertheless, continue to postulate the tic was confirmed by histopathologic participation of the oligodendroglial cell line in optic evaluation of each of the surgical specimens. nerve glioma.1 This viewpoint became more tenable Deparaffinized sections from all ten optic nerve glioma specimens were stained individually for glial fibrillary acidic protein (GFAP) with a polyclonal an- From the Departments of *Pathology and tDevelopmental Anat- tiserum raised in rabbits, HNK-1 which recognizes a omy and Neurosciences, Case Western Reserve University, Cleve- carbohydrate epitope in the myelin-associated glyco- land, Ohio, the ^Department of Ophthalmic Pathology, Armed 6 Forces Institute of Pathology, Washington, DC, and the §Depart- protein, A2B5 which recognizes a plasma membrane ments of Ophthalmology and Pathology, Emory University, At- antigen of ,7 vimentin (Sigma, St. Louis, lanta, Georgia. MO), S-100 (DAKO, Carpinteria, CA), and laminin Supported in part by an unrestricted departmental grant (HEG) (Bethesda Research Labs, Gaithersburg, MD). Five of from Research to Prevent Blindness, Inc. The opinions or assertions contained herein are the private views the optic nerve glioma specimens were stained for neu- of the authors and are not to be construed as official or as reflecting ron-specific enolase (NSE; DAKO), myelin basic pro- the views of the Department of the Army or the Department of tein (MBP; CalMed, San Francisco, CA), epithelial Defense. membrane antigen (EMA; Miles, Naperville, IL), ker- Submitted for publication: November 19,1990; accepted April 3, atin (EY, San Mateo, CA), and cytokeratin (Triton, 1991. 8 Reprint requests: Hans E. Grossniklaus, MD, L. F. Montgomery Alameda, CA) using a four-step method. Endoge- Eye Pathology Laboratory, Room 1603, Emory Eye Center, 1327 nous peroxidase activity was blocked by treating the Clifton Road NE, Atlanta, GA 30322. sections with methanol and hydrogen peroxide. The

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Table 1. Human optic nerve glioma place of the specific antiserum or by omitting the anti- immunostaining properties (number positive/ serum. All cellular elements were identified by histo- number examined) logic criteria.

Astrocytes Results Antibody N Perikarya Processes Oligodendrocytes The immunohistochemical staining characteristics GFAP 10 10/10 10/10 0/10 of optic nerve glioma are summarized in Table 1. The HNK-I 10 10/10 10/10 0/10 perikarya and processes of astrocytes in the optic A2B5 10 0/10 0/10 0/10 Vimentin JO 10/10 10/10 0/10 nerve gliomas (Fig. 1 A) were positive for GFAP. Anti- Laminin 10 0/10 0/10 0/10 vimentin reacted with neoplastic astrocytes in all of S-100 10 8/10 8/10 0/10 the optic nerve gliomas (Fig. IB). Antivimentin MBP 5 0/5 0/5 5/5 NSE 5 0/5 0/5 0/5 stained astroglial processes both intensely and dis- EMA 5 0/5 0/5 0/5 tinctly. However, reactivity for vimentin in astrocytes Keratin 5 0/5 0/5 0/5 was less prominent than staining with anti-GFAP. Cytokeratin 5 0/5 0/5 0/5 HNK-1 antisera was moderately reactive with astro- cytes in all gliomas (Fig. 1C). Astrocytes were negative for A2B5 in all glioma specimens. In all cases, laminin sections were incubated sequentially with 10% nor- antisera was unreactive with astrocytes, although con- mal goat serum followed by primary and secondary nective tissue elements such as dura, fibroblasts,an d antibodies. The sections then were incubated with the endothelial cells were moderately positive. The pro- appropriate species-specific peroxidase-antiperoxi- cesses and perikarya of astrocytes in eight often optic dase complex (1:10) and diaminobenzidine. Control nerve gliomas reacted weakly with S-100 antisera. In stains were done by applying preimmune serum in most specimens, astrocyte morphology was not well

Fig. 1. Staining is positive in perikarya and processes of neoplastic astrocytes for GFAP (A), vimentin (B), and HNK-1 (C). Linear arrangements of oligodendrocytes are positive for MBP with lack of staining in neoplastic astrocytes (D) {peroxidase-antiperoxidase, original magnifications A-C X40, insets XI60, D X10).

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delineated by anti-S-100. We found NSE was weakly suggests that the proliferating astrocytes in the optic positive in surviving axons in the optic nerve gliomas, nerve glioma () originate from but the neoplastic astrocytes were negative. Anti- the type 1 rather than from the type 2 astrocyte cell MBP stained surviving myelin sheaths in all optic line. Type 1 astrocytes appear to retain a greater capac- nerve gliomas evaluated with this reagent. Reaction ity for cell division than do type 2 astrocytes, and with MBP antisera revealed intensely stained linear most astrocytes that are stimulated to divide in re- patterns of cells whose histologic appearance was con- sponse to trauma in the adult sistent with that of nonneoplastic oligodendrocytes have a type 1 phenotype. In view of these observa- (Fig. ID). None of these cells reacted with anti-GFAP. tions, it is not surprising that the neoplastic astrocytes Astrocytes were negative for MBP. Antisera against of optic nerve gliomas appear to show a type 1 pheno- epithelial markers including EMA, cytokeratin, and type. keratin showed no positive staining in any of the optic At 15-19 weeks of fetal development, vimentin is nerve gliomas tested. the major intermediate filament protein, and GFAP is less prominent in glia of the human optic nerve.10 Discussion The expression of GFAP increases rapidly after 19 weeks, and the ratio of GFAP to vimentin increases The immunoreactivity for GFAP of astrocytes in until vimentin becomes a minor component in adult optic nerve gliomas (pilocytic astrocytomas) confirms tissue.10 In our study, the neoplastic astrocytes of op- the results of numerous earlier studies,9 and as ex- tic nerve pilocytic astrocytoma showed prominent vi- pected, GFAP was expressed strongly in perikarya mentin positivity. This is consistent with reports of and processes of an apparent majority of neoplastic prominent vimentin expression in astrocytomas.9 and reactive astrocytes in our study. The embryonic immunophenotypic pattern of the Further analysis of our results provides insight re- type 1 astrocyte (A2B5 negative and HNK-1 positive) garding the histogenesis of human optic nerve glioma. in conjunction with the prominent vimentin positiv- Experimental studies of gliogenesis in the rat optic ity present in the neoplastic astrocytes of our tumor nerve suggested that the adult nerve contains at least specimens strongly suggests that these histologically three distinct types of macroglial cells: oligodendro- well-differentiated tumors are expressing some imma- cytes, type 1 astrocytes, and type 2 astrocytes.4'5 Oli- ture antigens or are undergoing some degree of dedif- godendrocytes show surface labeling with anti-MBP ferentiation. but not with monoclonal anti-A2B5, and they have Malignant human glial cells were shown to express no intracellular reactivity for GFAP.45 Both type 1 laminin and EMA.911 Laminin is expressed by imma- and type 2 astrocytes label intracellularly with anti- ture and neoplastic astrocytes and malignant human GFAP antibodies; however, only the cell surfaces of glial cells in vitro.11 In vivo, however, only normal type 2 astrocytes label with anti-A2B5.4-5 In early de- perivascular astrocytes and nonreactive astrocytes at velopment, most of the immature type 1 astrocytes the brain surface contain laminin.11 In addition from the rat cortex contain HNK-1 as a cell-surface EMA-positive tumor cells were found in intracranial antigen. Later in development, HNK-1 expression malignant astrocytomas.9 In our study, laminin and becomes restricted to type 2 astrocytes. Types 1 and 2 epithelial markers, eg, EMA, cytokeratin, and kera- astrocytes differ in morphology, response to growth tin, were not detected in the neoplastic astrocytes of factors, and ontogeny.45 Type 1 astrocytes arise dur- optic nerve glioma. The lack of detectable expression ing early embryonic development from cells of the of these antigens in neoplastic astrocytes of optic optic stalk; type 2 astrocytes develop postnatally from nerve pilocytic astrocytomas is consistent with the a bipotential progenitor cell that has the capacity to well-differentiated appearance and noninvasive be- give rise to oligodendrocytes under various environ- havior of these tumors. mental conditions. Experimental studies suggest that The variable immunoreactivity for S-100 is consis- this bipotential progenitor cell is not a product of the tent with lack of specificity of this reagent reported in optic stalk but that these cells migrate into the optic other studies.9 A similar lack of specificity was re- nerve from the brain during development.4'9 ported for NSE antiserum; this did not react with the Neoplastic astrocytes of the optic nerve in our study neoplastic astrocytes in our study.9'12 did not react with anti-A2B5 but were labeled by anti- In summary, the immunophenotype of neoplastic HNK-1 antibodies. This immunohistochemical pat- astrocytes in human optic nerve glioma (pilocytic as- tern is consistent with that observed in developing rat trocytoma) was similar to the antigenic profile of im- type 1 astrocytes. If human gliogenesis follows a simi- mature type 1 astrocytes in the rat. Human and rat lar pattern to that observed in the rat, our finding of patterns of glial development in the optic nerve may an A2B5 negative HNK-1-positive labeling pattern be analogous.1314 If so, then our findings suggest that

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an early type 1 astrocyte is the proliferating cell of and K cells identified by a monoclonal antibody (HNK-1). J Immunol 127:1024, 1981. origin in optic nerve glioma. Whether the astrocyte 7. Eisenborth GS, Walsh FS, and Nirenberg M: Monoclonal anti- attained an early stage of development or underwent body to a plasma membrane antigen of neurons. Proc Natl dedifferentiation is unclear. Studies of gliogenesis in AcadSciUSA76:4913, 1979. human fetal optic nerve would be useful to evaluate 8. Sternberger LA: Immunocytochemistry. 3rd ed. New York, this thesis. John Wiley and Sons, 1986, pp. 32-89. 9. Perentes E and Rubenstein LJ: Recent application of immuno- Key words: optic nerve glioma, pilocytic astrocytoma, im- peroxidase in human neuro-oncology. Arch Pathol 111:796, munohistochemistry, type I astrocyte, type II astrocyte 1987. 10. Quitschke W, Sibony P, and Schechter N: Variable expression References of intermediate filament proteins during embryonic develop- ment of human optic nerve. Exp Neurol 90:204, 1985. 1. Russell DS and Rubenstein LJ: In Pathology of Tumours of the 11. McComb RD and Bigner DD: Immunolocalization of laminin Nervous System. 5th ed. Baltimore, Williams and Wilkins, in neoplasms of the central and peripheral nervous systems. J 1989, p. 372. Neuropathol Exp Neurol 44:242, 1985. 2. Borit A and Richardson EP: The biological and clinical behav- 12. Cras P, Martin JJ, and Gheuens S: Gamma-enolase and glial ior of pilocytic astrocytomas of the optic pathways. Brain fibrillary acid protein in nervous system tumors: An immuno- 105:161, 1982. histochemical study using specific monoclonal antibodies. 3. Spencer WH: Optic nerve. In Ophthalmic Pathology: An Atlas Acta Neuropathol (Berl) 75:377, 1988. and Textbook, Spencer WH, editor. Philadelphia, WB 13. Kennedy PGE, Watkins BA, Thomas DGT, and Noble MD: Saunders, 1986, p. 2429. Antigenic expression by cells derived from human gliomas 4. Miller RH, Ffrench-Constant C, and Raff MC: The macroglial does not correlate with morphologic classification. Brain cells of the rat optic nerve. Neuroscience 12:517, 1989. 109:1261, 1986. 5. Raff MC: Glial cell diversification in rat optic nerve. Science 14. Kennedy PGE and Fok-Seang J: Studies on the development, 12:517, 1989. antigenic phenotype and function of human glial cells in tissue 6. Abo T and Bolch CM: A differentiation antigen of human NK culture. Neuropathol App Neurobiol 13:327, 1987.

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