Human PATHOLOGY VOLUME 14
Monoclonal and Polyclonal Antibodies Against Neural Antigens: Diagnostic Applications for Studies of Central and Peripheral Nervous System Tumors JOHN Q. TROJANOWSKI, MD, PhD, AND VIRGINIA M-Y LEE,PhD Studies o f neural specific proteins (proteins restricted to neurons and gila) represent one o f the most active frontiers in the cell biology o f the nervous system. Antibodies that r e c o g n i z e such molecules are p o w e r f t t l r e a g e n t s f o r the i n v e s t i g a t i o n o f a wide range o f problems in neurobiology, such as celhtlar differentiation and specialization, cell migration, and c e l l - c e l l i n t e r a c t i o n ? -4 As e m p h a s i z e d in e a r l i e r reviews in this f o r u m , antibodies against tissue- and cell-specific antigens are nsefid diagnostic reagents that provide i m p o r t a n t i n f o r m a t i o n that cannot be obtained by use o f conventional histologic methods. ~'6 A l t h o u g h the n u m b e r o f newly r e c o g n i z e d n e u r a l antigens is increasing at an extraordinarily rapid rate, only a minority o f these will prove usefid for diagnostic studies o f tuntors o f the central and peripheral nervous system. T h e explosive i n c r e a s e in the n u m b e r o f new nervous system-specific proteins is, in part, a direct result o f the introduction o f techniques for the production o f monoclonal antibodies (MAs) by K o h l e r Received October 12, 1982, from the Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania. Revision accepted for publication November 30, 1982. Supported in part by NIH grants NS-16723 (Dr. Trojanowski), NS-16970, and NS-18616, and March of Dimes Birth Defects Foundation grant 1-826 (Dr. Lee). Dr. Trojanowski is the recipient of Teacher- Investigator Development award K07 NS00762 from the NINCDS. Address correspondence and reprint requests to Dr. Trojanowski: Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of PennsylvaniaSchool of Medicine, Johnson Pavilion/G2, Philadelphia, PA 19104.
and Milstein. z-4a-a As with any teclmical innovation, new is not always better, and tile advantages o f MAs must be considered in tile context o f their limitations. This review draws attention to factors e n t e r i n g into tile decision o f w h e t h e r to use MAs o r conventional antibodies (CAs) for tile diagnostic evaluation o f h u m a n nervous system tumors, and includes a brief survey o f those neural antigens that have been used with some success as " m a r k e r s " for such t u m o r s in diagnostic pathology.
MONOCLONAL VERSUS POLYCLONAL ANTIBODIES AS DIAGNOSTIC REAGENTS Monoclonal antibodies d i f f e r f r o m polyclonal antibodies o r CAs in that a given MA is the p r o d u c t o f a single clone o f B cells. Antibodies derived fi'om a single clone o f B cells will be o f the same i m m u n o globulin class and subclass and will bind to tile same antigenic d e t e r m i n a n t o r epitope with the same affinity. Since monoclones are derived fi'om the fi~sion o f a n t i g e n - p r i m e d B cells (having a finite life span) with immortal myeioma cells, the e n d - p r o d u c t o f a successful fusion is a hybridoma, which may be perpetuated in tissue ctthure and which secretes a single, or monoclonal, antibody. Tile p r i m a r y advantages o f MAs are thus t h e i r exquisite specificity a n d the long-term availability o f immtmochemicals that do not vary over time, which provides a m o r e specific and s t a n d a r d i z e d set o f reagents for i m m u n o h i s tochemical use. O t h e r p r o p e r t i e s o f MAs in comparison with GAs have recently been reviewed fi'om the perspective o f an immunologist and cell biologist. 8
Volume '14, No. 4 (April '1983)
T i l e limitations o f MAs fox" c e r t a i n types of-i,llmunologic studies have been outlined, 8 but little information is available r e g a r d i n g the relative advantages and disadvantages o f using MAs in conjunction with immunohistoclmmical m e t h o d s fox" diagnostic studies in pathology. An a s s e s s m e n t o f the p e r f o r m a n c e o f MAs against two neural antigens, myelin basic protein and netn'ofi]aments, in rat and h u m a n nervous system tissue has recently been c o m p l e t e d ? ~ It was f o u n d that some MAs against myelin basic protein and some MAs that recognized n e u r o f i l a m e n t s were m o r e sensitive than CAs. However, some were less sensitive than CAs. T h u s , the increased specificity associated with MAs was not obtained at tile e x p e n s e o f sensitivit)', and the MAs coukl be used with conventional imlnunolfistoclmmical m e t h o d s as effectively as CAs. Conceivably, a mixture o f a g r o u p o f MAs that recognize d i f f e r e n t epitopes o f the same protein could p r o d u c e an immunologic reagent that is m o r e sensitive than a single MA. Such a reagent would also be m o r e sensitive than CAs against the same protein, since CAs comprise a m i x t u r e o f antibodies o f varying specificities and affinities. T h e advantages o f MAs over CAs may justify the substitution o f MAs for CAs in diagnostic studies o f l m m a n neoplasms. However, this issue remains to be evaluated in detail. Certainly, additional comparative studies are needed. Despite tile lack o f information on this subject, the n a t u r e o f certain classes o f antigens may necessitate the use o f highly specific antibodies such as MAs. At present, a limitation o f the use o f MAs for tile diagnostic evaluation o f neural t u m o r s is the lack o f availability o f M As that recognize some o f tile neural antigens that are proving usefld as diagnostic markers, which are discussed below. This necessitates the c o m m i t m e n t o f m o r e resources to the p r o d u c t i o u and m a i n t e n a n c e o f MAs in a pathology laboratory than is generally required for CAs. Howe v e r , with the c u r r e n t i n t e n s e i n t e r e s t in MAs as p r o b e s in cell biology a n d as d i a g n o s t i c r e a g e n t s , these limitations should be o v e r c o m e soon.
Intermediate Filaments I n t e r m e d i a t e filaments are cytoplasmic filanmnts 10 nm in d i a m e t e r that are f o u n d in m a m m a l i a n cells. "a2 T h e y are cytoskeletal elements that have a d i a m e t e r intermediate between the diameters o f two o t h e r major cytoskeletal c o m p o n e n t s , i.e., micro filaments (6 nm) and microtnbules (24 nm). Although indistinguishable at tile ultrastructural level, the five different classes o f intermediate filaments are biochemically and immunologically distinct. T o a certain extent they are cell- o r tissue-specific. T h e s e classes o f intermediate filaments are 1) neurofilaments, f o u n d only in nem'ons; 2) glial filaments, f o u n d in glial cells; 3) desmin filaments, f o u n d in muscle; 4) keratin filaments, fouxld in epithelial cells; and 5) vimentin filaments, f o u n d in mesenchymal cells such as fibroblasts. V i m e n t i n filaments have been r e p o r t e d to coexist with some o t h e r classes o f intermediate filalnents in certain cells, and most cells in tissue culture will express vimentin regardless o f their histogenesis. Recent reports suggest that CAs tlmt recognize vimentin, desmin, o r keratin filaments are usefid for tim evaluation o f a variety o f non-lleural tumors. ~z Conventional antibodies against glial filaments have been extensively used as diagnostic reagents for studies o f central nervous system tumors. 13 T h e y discriminate astrocytic and e p e n d y m a l neoplasms fi'om o t h e r types o f p r i m a r y and metastatic brain tumors. Antibodies that recognize n e m o f i l a m e n t s and decorate normal n e u r o n s have not yet been exploited in the same way as antibodies against grim f l m n e n t , but r e c e n t w o r k with a n t i n e u r o f i l a m e n t MAs is encouraging. '4-~6 In one study, a n t i n e u r o f i l a m e n t MAs d e c o r a t e d n e o p l a s t i c cells in a variety o f n e u r a l t u m o r s such as ganglioneuronms, pheocln'omocytomas, and neuroblastonms. In general, differentiated nenral t u m o r s m o r e frequently contained i m m n n o reactive n e u r o f i l a m e n t , and n o n e o f the t u m o r s o f n o n - n e u r a l derivation, such as gliomas and meningiomas, contained neoplastic cells with i m m n n o r e a c t i v e neurofilament. ~5 I f anti-intermediate-filament antibodies are to be used for diagnostic purposes, they must be m o n o specific f o r o n l y o n e i n t e r m e d i a t e f i l a m e n t class. Monospecific antibodies can be p r e p a r e d f r o m CAs, but the p r o c e d u r e is laborious. T h e p r e p a r a t i o n o f monospecific a n t i - i n t e r m e d i a t e - f l a m e n t antibodies is particularly challenging, since it has been shown that all five classes o f i n t e r m e d i a t e filaments can s h a r e tim same antigenic d e t e r m i n a n t . T h u s , the p r o d u c tion o f MAs that recognize epitopes restricted to one class o f i n t e r m e d i a t e filaments would e n h a n c e tile diagnostic usefidness o f these filaments as markers in the types o f studies considered here. T h e distribution o f n e u r o f i l a m e n t s in a h u m a n p l m o c h r o l n o c y t o m a , d e m o n s t r a t e d by a neurofilament-specific MA, is shown in figure 1. By both immunofluorescence and peroxidase-antiperoxidase m e t h o d s , " tim a n t i n e u r o f i l a m e n t MA labeled tumolcells. Tim immunoreactive netu'ofilament was arranged primarily ill ball-like profiles similar to those observed
NEURAL ANTIGENS AS MARKERS OF CENTRAL A N D PERIPHERAL NERVOUS SYSTEM TUMORS
T h e r e are t h r e e categories o f neural antigens t h a t a p p e a r p r o m i s i n g as m a r k e r s ill d i a g n o s t i c studies: intermediate filaments, enolase isoenzymes, a n d S-100 p r o t e i n . A n u m b e r o f o t h e r n e r v o n s system-specific proteins that may prove to be relevant markers fox" neural t n m o r s have been described, lint there is currently insufficient information to evahmte their potential as such. ~-4"9 N o n e o f tlm neural antigens discussed h e r e are tumor-specific; rather, they are cell- or tissue-specific and thns provide i m p o r t a n t information on the d e g r e e o f differentiation o r tile histogenesis o f a given t u m o r . T h e y may also provide prognostic i n f o r m a t i o n , but this possibility has not been extensively e x p l o r e d . 282
MONOCLONAL AND POLYCLONAL ANTIBODIES TO NEURAL ANTIGENS (l'rojanowski & Lee)
FIGURE 1. Paraffin-embedded sections of normal rat brainstem (a and b] and h u m a n pheochromocyloma (c and d] stained with antineurofilament monoclonal antibodies [MAs] (a and r or control supernatant from parent myeloma ceils [b and d) by means of the peroxidase-antiperoxidase technique, a, The perikarya of normal brainstem neurons stain diffusely with antineurofilament MAs (large a r r o w h e a d ) , but some neurons contain little or no immunoreacfive neurofilament [small arrowhead). Axons (the positively stained linear profiles) with neurofilaments can also b e seen. [x600.] b, The control section was negative. [x600.] c, In the p h e o c h r o m o c y l e m a , tumor cells contain Immunoreactive neurofilamenls arranged in ball-like profiles [arrowheads) similar to those observed in a rat pheochromocytoma cell line) ~ [ x650.] d, The control section is negative. (x650.] All sections were counterstained with hematoxylin. The specificity of these MAs for neurofilament as well as tissue preparation, i m m u n o h i s t o c h e m i c a l procedures, a n d controls have b e e n d e s c r i b e d in d e t a i l elsewhere.m.,+-~6
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glycerate and phosphoenolpyruvate. Currently t e r m e d nenron-specific enolase, 14-3-2 protein is o n e o f the three homodinlers o f this intracytoplasmic enzyme. Neuron-specific enolase is the d i m e r comprising two 7 snbnnits. So-called liver enolase comprises two c+ subunits, and the enolase in muscle comprises two B subunits. T h e B subunit is not present as either a honaodimer o r a h e t e r o d i m e r in neural elements. T h e et-ct d i m e r is the most widely distributed form o f this e n z y m e a n d is r e f e r r e d to as n o n - n e u r o n a l enolase. In tile nervous systeln, n o n - n e n r o n a i enolase is f o u n d in glial cells (astrocytes, e p e n d y m a i cells, oligodendroglia, and Schwann cells) as well as in araclmoidal endothelial cells. Enolase may also occur as a h e t e r o d i m e r , and two hybrids are r e c o g n i z e d - an ct-fl hybrid and an a - y hybrid. T h e distribution o f these hybrid enolase isoenzymes is incompletely characterized. T h e tissue and cellular distribution o f the enolase isoenzymes, particularly neuron-specific enolase and n o n - n e u r o n a l enolase, has been intensively studied with CAs by use o f i m m u n o c y t o c h e n a i c a l m e t h ods. Lm"-~ AItl~ough n o n - n e u r o n a l enolase is widely distributed, neuron-specific enolase is c o n f i n e d to n e u r o n s and n e n r o e n d o c r i n e cells ( p a r a n e u r o n s , cells
in a clonal cell line (PC12 cells) derived f r o m a rat p h e o c h r o m o c y t o m a . ~s Since the staining o f neurofilaments with tiffs MA in n o r m a l n e u r o n s is diffusely cytoplasmic (fig. 1), t h e ball-like profiles may r e p r e sent abnormal neurofilaments associated with transf o r m e d t u m o r cells. Anti-intermediate-filament antibodies have the potential for exploitation as sensitive reagents for the diagnostic e v a l u a t i o n o f t u m o r s i n c l u d i n g n e u r a l tumors. Cross-reactivity is a significant problem, but it can be o v e r c o m e with the use o f MAs. Since the expression o f intermediate filantents is developmentally regulated and since the presence o f intermediate illaments in neoplastic cells may be abnormal, MAs that recognize individual classes o f i n t e r m e d i a t e filaments or individual subnnits o f a given class o f intermediate filaments may prove useful in the assessment o f t u m o r prognosis as well. Enolase Isoenzymes O n e o f the first neural specific proteins r e p o r t e d was 14-3-2 p r o t e i n ? '~9 It was later fotmd to be one o f a g r o u p o f isoenzymes in the glycolytic pathway that catalyzes the interconversion of 2-phospho-D283
Volume 44, No. 4 [April '1983}
o f tile diffuse n e u r o e n d o c r i n e system, APUD cells). N e u r o e n d o c r i n e cells d i f f e r fi'om nenrons, however, in t h a t t h e f o r m e r c o n t a i n b o t h n e u r o n - s p e c i f i c enolase and n o n - n e u r o n a l enolase, whereas n e u r o n s contain only neuron-specific enolase. T h e expression o f neuron-sl~ecific enolase by n e u r o n s , like that o f n e n r o f i h u n e n t s , is d e v e l o p m e n t a l l y r e g u l a t e d . T h e r e f o r e , nenron-specific enolase may serve as a differentiation m a r k e r as well as a n e u r o n a l marker. It is i m p o r t a n t to realize, however, that conventional anti-neuron-specific-enolase antibodies recognize the Y s u b u n i t r e g a r d l e s s o f w h e t h e r it o c c u r s in a h e t e r o d i m e r or in a b o m o d i m e r . This is an i m p o r t a n t point to consider when CAs specific for the a,/3, or Y subunits are used in immunolfistoclmnfical studies. In studies o f nervous system t u m o r s using CAs that recognize e i t h e r tile a, tile/3, o1" tlm y subunit, ~9--0~ only astrocytomas, e p e n d y m o m a s , oligodendroglionits, nleningiolnas, and schwannomas were shown to contain n o n - n e H r o n a l enolase. N o n e o f these tumors c o n t a i n e d n e u r o n - s p e c i f i c enolase, a n d it was not present in nmdtflloblastonaas o r cerebral neuroblastomas, h n m u n o r e a c t i v e / 3 subunit also was not f o u n d in any o f these tumors. Neuron-specific enolase has b e e n d e m o n s t r a t e d in l n e l a n o m a s a n d p e r i p h e r a l n e u r o b l a s t o m a s , h o w e v e r . 2~ A n o t h e r p r e l i m i n a r y study suggests that nenron-specifc enolase may be a m a r k e r f o r t u m o r s o f the d i f f u s e n e u r o e n d o c r i n e system (apudomas) as well. 19 Cross-reactivity does not a p p e a r to be a significant problem when CAs specific fin- ncnron-specific enolase, n o n - n e u r o n a l enolase, or t h e / 3 subunit are used as diagnostic reagents. Nevertheless, the |nab|lit}' to d i f f e r e n t i a t e the a - ' y hybrid fi'om n e u r o n specific enolase with CAs does present a problem in evaluating tile possible n e u r o n a l histogenesis o f a given n e o p l a s m . T h e p r o b l e m conld be p u r s u e d fin'tlmr by double labeling studies using a panel o f antienolase subtmit antibodies. A n t i b o d i e s t h a t r e c o g n i z e individual e n o l a s e snbtmits a p p e a r to be usefifl intmunochemicals for tim diagnostic a s s e s s m e n t o f I m m a n c e n t r a l a n d peripheral nervous system tumors. Tile inability to discriminate neuron-specific enolase fl'om tile a - y subunit remains a problem. Tile d e v e l o p m e n t o f MAs against individual enolase subunits may e n h a n c e their usefulness as markers in diagnostic pathology. Evid e n c e that tile expression o f tim 7 subunit is develo p m e n t a l l y r e g n l a t e d suggests that enolase isoenzymes may be exploited for assessing tile prognosis associated with such tumors.
neural proteins, its fimction has not yet been established u n e q u i v o c a l l y . Similarities to c a l m o d u l i n suggest tlmt it is one o f a g r o u p o f cMcitnn-binding proteins that includes t r o p o n i n C, parvalbnmin, and intestinal calcium-binding protein. ~-'-'-~Cttrrently, two biochelnically distinct forms o f S-100 protein are recognized: O n e form, S-100a, comprises cz a n d / 3 snbunits; the o t h e r fornt, S-100b, comprises only/3 subunits36 htitially considered to be restricted to cells o f tile central and p e r i p h e r a l nervous systems, S- 100 protein has also been d e m o n s t r a t e d in n e u r o e n d o c r i n e cells and, recently, in some n o n - n e u r a l cells. ~'~-'-'--~4'2GIn the c e n t r a l n e r v o u s s y s t e m it a p p e a r s i m m u n o h i s tochemically to be localized only in glial cells, althongll a neuronal localization has also been reported. In tile peril)heral nervous system, i m m u n o r e a c t i v e S-100 proteins in Schwann cells and satellite cells o f sympathetic ganglia as well as in glial elements o f tim enteric nervous system have been r e p o r t e d . In a n u m b e r o f studies, CAs against S-100 protein have been used for tile histogenetic classification o f htunan tumors. Astr0cytic neoplasms, oligodendrogliomas, neurofibronlas, and schwannomas contain i m m u n o r e a c t i v e S-100, whereas nteningiomas and medulloblastomas do not. Melanocyte-derived neoplasms such as melanomas and nevi also contain S-100 protein. Tile recent d e m o n s t r a t i o n o f S-100 p r o t e i n in g r a n u l a r cell t u m o r s by m e a n s o f tile peroxidase-antiperoxidase m e t h o d was cited in support o f the hypothesis that these tnnlors are derived fi'om tile glial elements o f the peripheral nervous system. T h e p r e s e n c e o f S-100 p r o t e i n in some nonn e u r a l cells a n d tile c o n t r o v e r s y s u r r o u n d i n g its localization in n e u r o n s indicates that conclnsions regarding tile histogenesis o f t u m o r s studied with CAs against this molecule must be tentative until m o r e information is available on its biodmmical features and celhflar distribution. At present, an S-100a and an S-100b protein are recognized; ftn'ther research ntay u n c o v e r o t h e r forms or snbtmits o f this protein. Conceivably, d i f f e r e n t forms o f S-100 protein with d i f f e r e n t cellular distributions, analogous to tile diff e r e n t forms o f enolase isoenzymes, will be found. Monoclonal antibodies that recognize d i f f e r e n t forms o f this protein could contribute to a better u n d e r standing o f its cell biologic features. Such MAs could also e n b a n c e tile usefulness o f S-100 protein as a diagnostic m a r k e r in pathology. However, a prelinfinary r e p o r t on tile use o f anti-S-100 MAs for t n m o r studies was not encouraging, zr
S-100 protein is an acidic protein that was discovered at the same time as neuyon-specific enolase, or 14-3-2, protein, m9 T h e desig'fiation S-I00 refers to tile solubility o f tiffs protein in a saturated solution o f a m m o n i n m sulfate at p H 7. It is f o u n d p r e d o m i nantly, but not exclusively, in elements o f tim nervons system and diffuse n e u r o e n d o c r i n e system. 1"2:-2-24 Although S-100 is one o f the most extensively studied 284
Tiffs b r i e f review has focused oi1 the use o f MAs and GAs that recognize neural antigens, as diagnostic reagents for studies of human nervous system tumors. Tile potential usefillness o f MAs against such antigens has been discussed in relationship to three
MONOCLONAL AND POLYCLONAL ANTIBODIESTO NEURAL ANTIGENS (lrojanowski & Lee)
categories o f nervous system-specific proteins (certain classes o f i n t e r m e d i a t e filaments, certain enolase isoenzymes, and S-I00 protein), whicit appear promising as diagnostic nmrkers for the evaluation o f neoplasms o f the peripheral and central nervous systems. T h e greater specificity o f MAs is a distinct advantage over CAs. Limitations in the use of MAs in diagnostic pathology are likely to be overcome in the near filture. Largely as a result o f the introduction o f laybridoma teclmology, antigens specific to nearly all m a j o r c a t e g o r i e s o f n e u r a l cells have b e e n reported. ~-4"9'~~ If MAs that recognize some o f these a n t i g e n s can be d e v e l o p e d f o r use in p a t h o l o g y laboratories, they will u n d o u b t e d l y improve diagnostic accuracy and contribute to o u r u n d e r s t a n d i n g of the histogenesis and cell biologic features of nervous system tumors.
I i. 12. 13.
14. 15. 16. 17.
1. Eng LF, Bigbee JW: Immunocytocllemistry of nervous systcm-spccific antigens. In Agranoff BW, Aprison ,tilt (eds): Advances in Neurocllemistry, vol 3. New York, l~lentnn Press, 1978 2. Langley OK, Ghandonr MS, Gombos G, et ah Monoclonal antibodies as nettral cell surface markers. Nettrochem Res 7:349, 1982 3. Mckay R, Raft MC, Reichardt LF (eds): Monoclonal Antibodies to Neural Antigens. Cold Spring Harbor Reports in the Neurosciences, vol 2. Cold Spring Harbor, Cold Spring Harbor Laboratory, 1981 4. Schaclmer M: Cell type-specific surface antigens in the mammalian nervous system. J Nenrochem 39:1, 1982 5. Pinkus GS: Diagnostic immunocytocltemistry of paraffinembedded tissues. Hunl Pathol 13:411, 1982 6. Taylor CR, Kledzik G: hnnmnolfistologic techniqttes in surgical pathology: a spectrum of "new" special atains. Hum Pathol 12:590, 1981 7. Kohler G, Milstein C: Continuous cuhnres of fltsed cells secreting antibody of pre-defined specificity. Nature 256:495, 1975 8. Edwards PAW: Some properties and applications of monoclonal antibodies. Biochem J 200:1, 1981 9. Mitchell MS, Octgen HF (eds): tlybridonms in Cancer Diagnosis and Treatment. Progress in Cancer Research and Therapy, vol 21. New York, Raven Press, 1982 10. Hickey WF, Lee V, Trojanowski JQ, et al: hnmtmohistochemical application of monoclonal antibodies against myelin basic protein and neurofilament triplet protein subunits:
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