1150 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / Augusr 1 Vol. 24

Ocular Tumors React with Anti-neuroblastoma Monoclonal Antibodies

Christione Helson ond Lawrence Helson

The reactivities of mouse monoclonal antibodies directed patibility complex, or a class I molecule unique to against human neuroblastoma and peripheral melanoma melanoma. We also tested the reactivity of a mono- associated antigens, with human retinoblastoma and cho- clonal antibody (DW-1) directed against a primitive roidal melanoma cell lines were tested. Segregation of an- neuroectodermal tumor cell line established from one tigenic determinants according to each tumor class and cell of the patients in our institution3'4 with these ocular line were observed. Three retinoblastoma cell lines and one fresh tumor explant showed determinants detected by two tumors. The monoclonal antibodies Q14 and R24 were obtained from A. H. Houghton of the Memorial human neuroblastoma antisera, while the choroidal mela- 5 noma showed one determinant present on a peripheral mel- Sloan-Kettering Cancer Center. anoma but not neuroblastomas nor retinoblastomas, sug- The tumor cell line SK-RB-DU was derived from gesting certain potential distinctive tumor related deter- a previously untreated patient with bilateral retino- minants. Invest Ophthalmol Vis Sci 24:1150-1152, 1983 blastoma.6 The patient's tumor was initially passaged in nude mice and then later transferred to cell culture. The application of monoclonal antibody technol- The cell line growing as a monolayer was passaged ogy permits the analysis of the phenotypic hetero- continuously for over 50 subtransfers during one geneity of both normal and tumor tissues. This is year. The SK-RB-MI and SK-RB-SP lines were also based upon the observation that hybridoma-derived derived from patients with bilateral retinoblastoma. monoclonal antibodies can be used to detect specific These were initially cultured in the presence of a fi- antigenic determinants in cells. Although the func- broblast feeder cell line derived from the conjunctiva tion and importance of these determinants are gen- of another retinoblastoma patient. These cells tend erally unknown, knowledge of their distribution to remain as clumps of 30 or 40 cells loosely attached among tumors may have utility as a complementary to the fibroblast monolayer and are readily harvested technique to light and electron microscopy for char- by simple agitation and collection. The tumor cells acterizing different tumor cell phenotypes and pos- grow poorly or not at all in the absence of these feeder sibly for resolving the complex interrelationships be- fibroblasts, and attempts at culture with normal skin tween tumor cells of even the same type. We decided fibroblasts or autologous tumor associated fibroblasts to use monoclonal antibodies to define more clearly were consistantly unsuccessful. These cell lines were the relationship of eye tumors and other tumors com- tested for reactivity with monoclonal antibodies prior monly considered to be analogs eg, retinoblastoma: to and after short-term culture. Retinoblastoma tu- neuroblastoma and choroidal: peripheral melanoma. mor tissue, SK-RB-Ne, was obtained with Vi hr after We screened the reactivities of three retinoblastoma enucleation of a tumored eye from an untreated pa- and one ocular melanoma cell lines with eight mono- tient with bilateral retinoblastoma. Tumor imprints clonal antibodies raised against antigens of different on polylysine-treated glass slides were made, air dried, tissues. The latter include fetal brain, neuroblastoma and fixed and stained as described below. This same cells, melanoma cells, and primitive neuroectodermal tumor is currently in short-term culture and it is too tumor. early to consider it an established cell line. The ocular Materials and Methods. Six monoclonal antibod- melanoma cell line SK-OM-FO was obtained from ies were obtained from J. T. Kemshead.1 An addi- an untreated patient and maintained in vitro for 8 tional antineuroblastoma monoclonal antibody BRL- months prior to antibody testing. The peripheral 7982 AS was obtained from a commercial source melanoma cell line RPMI7931, originally established (Bethesda Research Laboratories, Gaithersburg, MD). by G. Moore in Rosewell Park Memorial Institute, This antibody is widely reactive with neuroectoder- New York, was obtained from Jorgen Fogh of Me- mal tumor cells and appears to recognize a cell surface morial Sloan-Kettering Cancer Center and main- glycoprotein of Mr = 2000 on the human neuroblas- tained in our laboratory since 1975. The human neu- toma cell line IMR-5. This cell surface determinant roblastoma cell line SK-N-MC was included in this is present on human fetal brain cells, pre-B cells, study for comparison. It is a well-established cholin- mature B cells, and their corresponding leukemias.2 ergic neuroblastoma cell line and is one of the two The antibody does not recognize foreign fibroblasts, neuroblastoma cell lines of eight tested that did not or two of eight human neuroblastoma cell lines in react with the UJ13A monoclonal antibody. It is also our laboratory, indicating that the antigenic deter- distinctive in that it is only one of eight human neu- minant it does recognize is not of a major histocom- roblastoma cell lines that contained a determinant

0146-0404/83/0800/1150/S0.95 © Association for Research in Vision and Ophthalmology

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Table 1. Cell line Peripheral Retinoblastoma Choroidal melanoma Monoclonal Neuroblastoma melanoma RPMI antibody SK-N-MC SK-RB-DU SK-RB-MI SK-RB-SP SK-RB-NE SK-OM-FO 7931

Antineuroblastoma BRL-7920 AS UJ13A UJ181.4 UJ127:11 UJ3O8 UJ223.8 UJ167 DW-1

Antimelanoma Q14 nd nd R24 nd nd

recognized by UJ181.4. These latter studies were tumor antibody DW-1. Within this group of tumors, done using conditions identical to those described in the ocular melanoma is distinguishable from the pe- this study for these tumor lines. ripheral melanoma by the reactivity with the R24 The cells were assayed for reactivity to antibody antibody (Table 1). using an indirect fluorescent method. The tumor cells Discussion. The BRL-7920 AS recognized a prod- were permitted to attach and grow as a monolayer uct present in most of these cells tested. In additional on a LAB-TEK® plate. (Miles Laboratories, Naper- studies with this antibody, it did not recognize two ville, IL). They were then fixed with ice cold methyl out of eight neuroblastoma cell lines. In view of the alcohol and then washed with cold phosphate buff- information known about this antibody, it does not ered saline pH 7.4 (PBS). They were then exposed for seem probable that it is recognizing HLA-D/DR de- 45 min at 4 C to antihuman neural tissue antibodies. terminants or a molecule specific to these tumors. After two additional washes with PBS, fluorescinated Since the product being recognized appears to be dis- goat antimouse IgG or IgM (Meloy Labs., Gaithers- tributed among a variety of cell classes including lym- burg, VA) was applied for 1 hr at 4 C. After thorough phocytes, the use of this antibody for classification washing with PBS, the cells were mounted in cold 1% of these ocular tumors is probably of little value. glycine in water at a pH 7.2 and fluorescence visually UJ13A is an antibody raised against human fetal scored using a halogen epifluorescent light source on brain tissue. It recognizes a surface glycoprotein in a Nikon microscope. Visual scoring was registered as many tumors of neural origin. These include neu- positive (+) when bright green fluorescence was ob- roblastomas at a variety of levels of differentiation. served at the lowest antibody dilution and this same This determinant found in the choroidal melanoma dilution of antibody stained cells using the vectastain™ and the four retinoblastomas suggest these tumors to ABC Kit, with biotinylated antimouse IgG (Vector 7 be less differentiated than the peripheral melanoma, Labs., Burlingame, CA). or the cholinergic neuroblastoma, both of which were In each experiment, the fluorescinated or bioti- derived from older patients. The SK-N-MC neuro- nylated antimouse antibody added to replicate cells blastoma not only did not exhibit the particular an- without monoclonal antibody served as controls. In tigenic determinant recognized by UJ13A but was all cases the absence of fluorescence in controls was unique among neuroblastomas because it also ex- obligatory to exclude nonspecific absorption. pressed an antigen recognized by UJ181.4. In a per- Results. The BRL-7920 AS antibody was reactive sonal communication, J. T. Kemshead reported that with determinants on both melanoma, 3/5 retino- the UJ181.4 was restricted to fetal brain, neuroblas- blastoma and 6/8 neuroblastoma cell lines tested. The toma, medulloblastoma, and retinoblastoma. This cells of ocular origin in this study contained an an- antibody did detect determinants in four retinoblas- tigen recognized by UJ13A, while the UJ181.4 may toma cell lines, suggesting that in addition to the pan- be recognizing an antigen peculiar to this one unusual neuroectodermal antigen seen by UJ13A, the reti- neuroblastoma, and 4/4 retinoblastoma cells. This noblastoma exhibit fetal antigens that may be also notion of segregation of determinants was reinforced found in neuroblastomas or other neuroectodermal by the selective reactivity of antimelanoma antibody tissues. Q14, R24, and the antiprimitive neuroectodermal The DW-1 antibody, raised against a primitive

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neuroectodermal tumor antigen was reactive with five patients with bilateral disease and may not be both the choroidal and the peripheral melanoma, sug- expressed by unilateral retinoblastomas. The antigens gesting the presence of an additional antigen in these are probably not tumor specific but may represent cells which is separate from the "fetal" product rec- different stages of differentiation. By analyzing anti- ognized by UJ13A. genic determinants using a battery of monoclonal In comparing the reactivity of the anti-melanoma antibodies of more cell lines and tumors from a wider antibodies with other antibodies, we were not sur- variety of patients, a more comprehensive perspective prised to find a degree of specificity since these former of these tumors may be possible. antibodies may be recognizing class I molecules; dis- Key words, ocular tumors, monoclonal antibodies tinguishing yet another set of antigens. Similar reactivity among these tumors of rather Acknowledgments. The authors are indebted to Dr. Rob- disparate origin, ie, infants with neuroblastoma and ert Ellsworth of the Ophthalmology Department, New York adults with neuroblastoma or melanoma, is not un- Hospital, New York, for tumor material; Drs. J. T. Kems- expected. In fact, reactivity of neuroblastoma, reti- head of the Imperial Cancer Research Fund, Institute Child noblastoma, and melanoma cells with a single mono- Health, 3rd Floor, Guilford Street, London WC1N 1EH, 8 UK, and A Houghton of Memorial Sloan-Kettering Cancer clonal antibody has already been reported. What Center, New York, for their generous gifts of Neuroblas- may eventually have more clinical significance is the toma and Melanoma antisera, respectively. distribution among cells and the biochemical iden- tification of the specific determinants being recog- From the Pediatrics Research Laboratory, Memorial Sloan-Ket- tering Cancer Center, New York, New York. Supported in part by nized by these monoclonal antibodies. the ACS-PDT #186 Grant and the Anne Marie O'Brien Fund. The data suggest that there may be fundamental Submitted for publication: September 7, 1982. Reprint requests: antigens common to most neuroectodermal tissues Dr. Lawrence Helson, Head, Pediatric Cancer Research Lab., or tumors that are independent of the state of differ- Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, entiation such as that recognized by BRL-7920 AS. New York, NY 10021. There may also be a variety of differentiation antigens exhibited potentially by all neuroectodermal tissues References but whose expression is variable. 1. Kemshead JT, Fritschy JG, Asser U, Brown S, and Greaves If the UJ 13 A and the UJ 181.4 antibodies recognize MF: Characterisation of an antigen present on neuroblastoma "fetal" antigenic substances then this suggests the and melanoma cells recognized by monoclonal antibody UJ presence of lesser differentiated status for the reti- 127:11. In Pediatric Oncology. Proceedings of the XIII Meet- noblastoma and choroidal melanoma tumor types ing of the International Society of Pediatric Oncology, Mar- than the cholinergic neuroblastoma and peripheral seilles, 1981, Raybaud C, Clement R, Libreuil G, and Bernard JL, editors. Amsterdam, Excerpta Medica, 1982, pp. 85-89. melanoma. 2. Kennet RH, Jonak Z., and Bechtol KB: Characterization of The perspective of the primitive neuroectodermal antigens with monoclonal antibodies. In Advances in neuro- tumor as a nosologic entity is based upon histologic blastoma Research. Evans AE, editor. New York, Raven Press, criteria that assumes it to be a preneuroblastoma. The 1980, pp. 205-219. reactivities observed with DW-1 suggest that there are 3. Helson L, Helson C, Lieberman P, and Schneider B: Primitive neuroectodermal tumors (PNET). Proc. XVII Annual Meeting additional antigens being exhibited in the melanomas of Am Soc Clin Oncol April 30-May 2, 1981. Abstract #C- that are of a less differentiated nature, but distinct 14, p. 336. from those found in fetal central nervous system tis- 4. Kohler G, and Milstein C: Derivation of specific antibody- sues. producing tissue culture and tumor lines by cell fusion. Eur In conclusion, ocular tumors may not be very J Immunol 6:511, 1976. 5. Dippold WG, Lloyd KO, Li LTC, Ikeda H, Oettgen HF, and unique compared to other neuroectodermal tumors Old LJ. Cell surface antigens of human malignant melanoma; in terms of expression of antigenic determinants. The definition of six antigenic systems with mouse monoclonal retinoblastomas appear to retain what may be two antibodies. Proc Natl Acad Sci USA 77:6114, 1980. "fetal"-type antigenic products that can be detected 6. Helson L, Kitchin FD, Abramson D, and Ellsworth RM: Metastatic retinoblastoma and L-phenylalanine mustard-dian- by two different monoclonal antibodies. The retino- hydrogalactitol. Anticancer Res 2:235, 1982. blastomas may be distinguished from the melanomas 7. Hsu SM, Raine L, and Fanger H: A comparative study of the in that the latter appear to express both fetal (UJ 13 A, peroxidase-antiperoxidase method and an avidin-biotin com- DW-1) and antigens associated with more differen- plex method for studying polypeptide hormones with radioim- tiated tissue and recognized by Q14 and R24. The munoassay antibodies. Am J Clin Pathol 75:734, 1981. antigenic substances defined by the UJ 181.4 mono- 8. Kemshead JT, Fritschy J, Asser U, Sutherland R, and Greaves MF: Monoclonal antibodies defining markers with apparent clonal antibody in human retinoblastoma cells ap- selectivity for particular haemopoietic cell types may also de- pear to have rather a unique pattern of distribution. tect antigens on cells of neural crest origin. Hybridoma 1:109, This is based only on its reactivity in five tumors from 1982.

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