Review: Monoclonal Antibodies and the Skin Biopsy: Current and Potential Clinical Applications

Review: Monoclonal Antibodies and the Skin Biopsy: Current and Potential Clinical Applications BY JO-DAVID FINE, MD

ABSTRACT: Monoclonal antibodies are becoming increasingly useful in the clinical diagnosis and/or treatment of a number of unrelated diseases. Discussion will be directed to those monoclonal antibodies recognizing antigens within the skin which appear to have either proven or potential application in the diagnostic evaluation of the skin biopsy. KEY INDEXING TERMS: Monoclonal antibodies; Intermediate filaments; Basement membrane; Tumor antigens; Epidermolysis bullosa. [Am J Med Sci 1985; 290(4):143-151.]

O

ver the past few years, increasing numbers of laboratories have been successful in producing monoclonal antibodies to a variety of structural antigens in normal and diseased tissues as well as to microorganisms, hormones, and enzymes. Originally utilized as immunohistochemical probes in basic research, several of these monoclonal antibodies have been used more recently for diagnostic therapeutic purposes. Using this technology, a wealth of new information has been generated regarding the structure From the Department of Dermatology, Uniuersity of Alabama at Birmingham School of Medicine, and Birmingham V/)terans Administration Medical Center; Birmingham, Alabama. Reprint requests .' Jo-Dauid Fine, MD, Department of Dermatology, Uniuersity of Alabama at Birmingham, Uniuersity Station, Box 76, Birmingham, Alabama 35294.

and function of various components of the skin both in health and in disease. As will be discussed, monoclonal antibodies are now available which have binding specificity for cell types within the epidermis (keratinocyte, melanocyte, Langerhans cell, Merkel cell) and dermis (fibroblasts, endothelial cells, appendageal structures), components of skin basement membrane Oaminin, type N collagen, fibronectin, KF-1 antigen, epidermolysis bullosa acquisita antigen, anchoring fibrils), and non-cellular elements within the dermis (types I, III, V, and VI collagen). Alterations in the expression (ie, KF-1, anchoring fibril-associated antigens) or immunogenicity (ie, epidermolysis bullosa acquisita antigen) of several of these antigens have been associated with specific disease states. Other antigens with as yet unknown functions have been defined within the epidermis of normal, ie, KF-2, and psoriatic skin. In addition, many tumorassociated antigens have been defined using monoclonal antibody technique. In this review, primary emphasis will be placed on the discussion of those monoclonal antibodies which have current or potential applications for diagnosis of biopsied skin. Discussion of the basic principles and methodology of monoclonal antibody production is beyond the scope of this article; interested readers are encouraged to refer to more comprehensive

THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

reviews on this subject. I - 3 Monoclonal Antibodies and the Epidermis Keratins. Keratins comprise a family of at least 19 different proteins which form intermediate filaments in epidermis and other epithelium; as such, keratins function as cytoskeleton in such tissues.4 These proteins have a range of molecular weights (40-70 kd) and pI values. Recently a number of different monoclonal antibodies have been produced which bind to some but not all keratins. On the basis of studies with three of these antibodies (AE1, AE2, AE3), it is now recognized that the presence of specific keratin species can be correlated with the degree of epithelial differentiation (simple vs. stratified vs. keratinized).5-8 When these monoclonal antibodies were applied to normal human skin, three different staining patterns were noted, dependent upon the keratin species recognized by each antibody. For example, AE1 monoclonal antibody binds to keratins with molecular weights of 50 and 56.5 kd; by indirect immunofluorescence, only basalar (most germinative) keratinocytes are stained. While both AE2 and AE3 monoclonal antibodies bind to keratins with molecular weights of 65-67 kd, AE2 and AE3 also bind to species with molecular weights of 56.5 and 58 kd, respectively. As a correlate, AE3 binds to all keratinocytes within normal epider-

143

Monoclonal Antibodies and Skin Biopsy

mis whereas AE2 does not bind to basalar keratinocytes. Studies have shown that keratinocytes in diseased skin may have altered staining by these monoclonal antibodies and therefore altered expression of keratin subclasses. For example, AE1 normally binds exclusively to basalar keratinocytes, whereas in several conditions ie, psoriasis, seborrheic dermatitis, actinic keratosis, verrucae, and in situ and invasive skin cancer, suprabasalar staining may be noted. 9 In contrast, another anti-keratin monoclonal antibody, K1, which normally binds to keratinocyte cytoplasm within the upper epidermis, binds to basalar keratinocytes in verrucae,lo Although the above pathological conditions are usually easily differentiated on the basis of well defined histological criteria, it is clear that each can be distinguished from normal skin using anti-keratin monoclonal antibodies as immunohistochemical stains. As a practical application, for example, anti-keratin monoclonal antibodies may be useful in determining the presence of residual skin cancer within the margins of excision at the time of surgery if frozen sections are obtained (by either conventional or microscopically-controlled chemosurgical techniques).ll Similarly, when the cell type of origin, ie, keratinocyte vs. melanocyte, of an anaplastic skin tumor cannot be determined by routine light microscopic criteria, additional information may be obtained by staining further tissue sections with a series of monoclonal antibodies directed against several different melanoma-associated antigens and keratins. In such a manner, 37/38 anaplastic tumors were recently successfully classified, thereby providing valuable information to the clinician as to both the size of surgical margins necessary at the time of definitive re-excision and the possible longterm prognosis .12 Similarly,

144

131/131 carcinomas, regardless of the degree of differentiation, were shown to express cytokeratins when monoclonal antibodies were used as immunohistochemical probes. 4 In another series, only 11/173 epithelial-derived tumors (metastatic prostatic adenocarcinoma, acinic cell tumor of the parotid, renal cell carcinoma) were not found to express cytokeratins; further studies will be needed to determine the potential diagnostic usefulness of the latter findings when evaluating metastatic lesions of possible prostatic or renal origin. 13 Recent 2-dimensional gel electrophoresis studies have shown that various carcinomas may be further subtyped on the basis of differences in expression of individual keratin proteins. 14 ,15 For example, adenocarcinomas from various organs express cytokeratins 8, 18, and 19, whereas squamous cell carcinomas lack these three keratins. Therefore, development of a series of monoclonal antibodies capable of distinguishing among the 19 keratin proteins should prove invaluable in further determining the tissue of origin of a cytokeratin-containing anaplastic lesion metastatic to the skin. In this manner, using monoclonal antibody CK-4 which is specific for cytokeratin 18, cells of extramammary Paget's disease have been distinguished from surrounding normal keratinocytes. 16 Additionally, although as yet unproven, it may be also possible in some situations to distinguish among some of the inflammatory skin conditions, such as psoriasiform eruptions, when less distinctive histological features are present. Profilaggrin and Filaggrin. Recently a monoclonal antibody was described that has specificity for profilaggrin, a high molecular weight (>200 kd) histidine-rich protein localized to keratohyaline granules within an upper region of the epidermis, the stratum granulosumP A breakdown product of

this protein, filaggrin, appears to be important in the induction of macrofibril formation by keratin intermediate filaments. Since the stratum granulosum is characteristically absent in psoriasis but present in other psoriasiform eruptions (including lupus erythematosus, pityriasis rubra pilaris, sebborheic dermatitis, and eczematous dermatitis), this monoclonal antibody should be of potentially great diagnostic usefulness in cases where psoriasis can not be easily differentiated from other conditions by routine histology. Similarly, the stratum granulosum may be absent in some cases of ichthyosis vulgaris. Since it may be frequently difficult to differentiate among the various types ofichthyosis clinically and histologically, demonstration of the absence of the stratum granulosum with this monoclonal antibody would be an important diagnostic finding; similar findings have recently been reported with polyclonal antibodies to filaggrin.1B In contrast, the stratum granulosum is characteristically markedly thickened in lichen planus. Although the latter disease is usually easily diagnosed by routine histological stains, such a monoclonal antibody may be helpful in the evaluation of difficult cases. Desmosomal Antigens. Several monoclonal antibodies have been described that bind in part or exclusively to desmosomes, including antibodies to desmoglein I, KF-2, and HK1 antigens. 19-21 In addition, polyclonal antibodies have been prepared against -desmoplakin (desmosomal plaque protein), which is expressed in normal epithelium and in tumors of epithelial origin.22 Such antibodies potentially may be useful in the evaluation of anaplastic tumors; for example, positive staining would suggest a tumor of epithelial origin rather than melanoma. Other Keratinocyte-Related Markers. Recently a monoclonal October 1985 Volume 290 Number 4

Fine

antibody was defined which recognizes keratinocytes in lesions of psoriasis but not within normal uninvolved skin.23 Subsequently this antibody has been shown to recognize keratinocyte antigens within other cutaneous conditions, including squamous cell carcinoma, keratoacanthoma, pityriasis rubra pilaris, irritated seborrheic keratosis, condyloma accuminatum, and lichen planus. 24 Interestingly this monoclonal antibody does not stain basal cell carcinoma. Non-Keratinocyte Cell Markers. Langerhans cells may be detected using commercially available monoclonal antibodies to either Ia antigen or the thymocyte cell surface marker, OKT_6. 25 .26 In addition, a new monoclonal antibody, Lag, has been recently described that has specificity for an extramembranous component of the Birbeck granule, an organelle found only in Langerhans cells. 27 However, with the exception of the recent demonstration of reduced numbers of Langerhans cells in skin from patients with AIDS, the study of skin biopsy specimens with these monoclonal antibodies has not as yet been shown to have diagnostic, prognostic, or therapeutic significance. 28 A human monoclonal cold agglutinin antibody recently has been described which binds exclusively to Merkel cells within the epidermis. 29 Unfortunately this antibody does not bind to Merkel cell carcinomas, thereby preventing its application in the diagnosis of this rare skin tumor. Merkel cells may also be demonstrated by their expression of cytokeratins. 30 A monoclonal antibody with binding specificity for melanocytes also has been reported .31 Such an antibody may be theoretically useful in the examination of skin specimens taken from areas of suspected vitiligo. Since melanocytes are absent in vitiliginous skin, no staining

..

'" '"---. ~';~ ' ''' L"

t·

I

I

"~ .',

'

.:.,,'.'

h

........D

,,~ .'

t, " [ , ' • . .

.~ t

....'l (~

'"'i::tP':,r

Figure 1. Electron micrograph of the skin basement membrane zone; reduced numbers of anchoring fibrils are present since this specimen was obtained from a patient with dystrophic epidermolysis bullosa (bar = 0.25 microns). E = epidermis; D = dermis. Asterisks = lamina luclda. Solid arrows = Lamina dense; open arrows = hemldesmosomes; arrowheads = anchoring fibrils.

should be seen with this antibody. In contrast, some staining should still be detectable in biopsies taken from patients with postinflammatory hypopigmentation clinically similar to or indistinguishable from vitiligo. Similarly, since some anti-melanoma monoclonal antibodies cross-react with normal melanocytes, these latter antibodies should also be useful diagnostically in the evaluation of vitiliginous skin. 32 Alternatively, anti-vim en tin monoclonal antibodies can be utilized since both melanocytes and Langerhans cells contain this type of intermediate filament instead of keratin. 33 Monoclonal Antibodies and Skin Basement Membrane Zone The dermo-epidermal junction, or basement membrane, is a critical structural and functional region of the skin. This basement membrane is composed of two portions, the lamina lucida and the lamina densa, each containing a number of distinct antigens (Figure 1). Laminin and Type IV Collagen. Polyclonal and monoclonal antibodies are now available against laminin, a high molecular

THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

weight (440 and 220 kd) glycoprotein component of the lamina lucida of the dermo-epidermal junction and other basement membranes. 34 Similarly, polyclonal and monoclonal antibodies have been prepared against type IV collagen, a component of the lamina densa of all basement membranes . 35 As will be discussed in greater detail later, antibodies to these two antigens are now routinely used diagnostically in the classification of patients with epidermolysis bullosa. Epidermolysis Bullosa Acquisita Antigen (EBA). A monoclonal antibody has recently been produced against the EBA antigen, a high molecular weight component (145 and 290 kd) of the lamina densa ofthe dermo-epidermal junction of primarily stratified squamous epithelium. 36 Since this antigen is relatively well expressed in skin from patients with all forms of congenital epidermolysis bullosa, it can be effectively substituted for type IV collagen in immunofluorescence mapping studies. 37 KF-1 Antigen. Using monoclonal antibody technique, a new primate- and stratified squamous epithelial-specific antigen, KF-1, 145

Monoclonal Antibodies and Skin Biopsy

was recently defined within the lamina densa of the dermo-epidermaljunction. 38 This antigen has a molecular weight of approximately 72 kd and is produced by human epithelioid cells in vitro.39 Although detectable in seemingly identical amounts in normal human skin and skin from patients with the epidermolytic (simplex) and junctional forms of epidermolysis bullosa, this antigen is present in reduced amounts or is absent in clinically-uninvolved skin from patients with dominant and recessive forms of dystrophic epidermolysis bullosa, respectively.40 These latter findings suggest a role for KF-1 antigen in the maintenance of dermoepidermal structural integrity and its importance in the marked skin fragility and sub-lamina densa blister formation found in patients with both forms of dystrophic epidermolysis bullosa. These findings also suggest possible diagnostic applications. For example, the lack of KF-1 antigen expression in skin from a patient previously shown by immunofluorescence mapping studies to have dystrophic epidermolysis bullosa is more compatible with recessive than dominant disease. This information may prove to be especially useful in the evaluation of newborns lacking positive family histories since some patients with dominant dystrophic epidermolysis bullosa are spontaneous mutations for the disease. Furthermore, since KF-1 antigen is detectable in normal fetal skin by the 16th gestational week, this monoclonal antibody may be potentially useful in the prenatal diagnosis of recessive dystrophic epidermolysis bullosa. 41 Anchoring Fibril-Associated Antigens. Two monoclonal antibodies (AF-1, AF-2) have been described with binding specificity for anchoring fibrils and, to a lesser extent, for the lower portion of the lamina densa. 42 These anti'bodies are primate- and stratified squamous epithelial-specific. Both 146

antigens appear to be normally expressed along the dermo-epidermal junction of normal human skin and skin from patients with simplex,junctional, and dominant dystrophic forms of epidermolysis bullosa. In contrast, neither anchoring fibril-associated antigen is detectable in skin from patients with the recessive form of dystrophic epidermolysis bullosa, consistent with the virtual absence of anchoring fibrils reported in such skin. Although each of these antibodies, therefore, appears to have diagnostic usefulness in identifying patients with recessive dystrophic epidermolysis bullosa, neither can be used for prenatal diagnosis since these anchoring fibril-associated antigens are not detectable until approximately 26 weeks of gestational age. 43 Furthermore, use of these antibodies alone may not distinguish between specimens from patients with junctional and dominant dystrophic forms of epidermolysis bullosa in contrast to use of KF-l. Immunofluorescence Mapping. 44 ,45 It has been well established by electron microscopy that skin cleavage or blister formation develops in distinct ultrastructurallocations within skin lesions of patients with each of the three inherited forms of epidermolysis bullosa. 46 ,47 In the epidermolytic form, epidermolysis bullosa simplex, blisters are intraepidermal, whereas in the junctional and dystrophic forms, blisters develop within the lamina lucida or beneath the lamina densa of the dermo-epidermal j unction, respectively. Since several well characterized basement membrane antigens have been localized exclusively to either the lamina lucid a or lamina densa, use of polyclonal or monoclonal antibodies to three of these antigens (bullous pemphigoid antigen, laminin, and type IV collagen) on a skin biopsy of a freshly induced blister will permit rapid and accurate determination of the level of

skin cleavage and therefore, the correct type of epidermolysis bullosa. This information is vital since the classification of the type of epidermolysis bullosa permits determination of course, prognosis, and therapy, as well as genetic counseling. In many laboratories this technique, referred to as immunofluorescence mapping, has now circumvented the need for performance of electron microscopy for the diagnosis of such patients. As previously noted, monoclonal antibodies to other well defined basement membrane antigens, such as the EBA antigen, can also be used in these studies. Monoclonal Antibodies and the Dermis

Collagens. At least eight biochemically and immunologically distinct types of collagen have been defined. 48 Of these, types I and III comprise the majority of the extracellular portion of the dermis. Minute amounts of types V, VI, and probably VII collagen can also be found in addition to types I, III, and IV (the latter basement membrane-associated). Very recently monoclonal antibodies have been generated to several of these collagen types. 49 As a result of the latter, distinct differences in the dermal localization of collagen types have been appreciated. Although still purely research immunoreagents, these monoclonal antibodies may possibly find diagnostic application in the demonstration of subtle differences in the distribution of collagen types in diseases associated with progressive changes in dermal collagen, such as scleroderma. Other Intermediate Filaments. 4,50 Vimentin. Monoclonal antibodies are now available which recognize vimentin, the 57 kd intermediate filament found in mesenchymally-derived cells such as fibroblasts, endothelial cells, and macrophages. Since neural tissues contain different types of intermediate filaments, it may be theoretically possible to differentiOctober 1985 Volume 290 Number 4

Fine

ate better between spindle tumors of the dermis derived from fibroblasts, ie, dermatofibroma, and those of neural origin. Similarly, as will be discussed later, other skin tumors may be identified by their expression of vimentin and/ or desmin. Desmin. Monoclonal antibodies have been prepared against desmin, the 53 kd intermediate filament expressed within normal tissue and tumors containing smooth and skeletal muscle elements. Endothelial cell antigens. Endothelial cells express vim entin and Factor VIII. 51 As such, monoclonal antibodies to either of these antigens, or to other endothelial-associated antigens, can be utilized to confirm the vascular nature of selected dermal tumors, including both classical and AIDSrelated Kaposi's sarcoma. Dermal glandular antigens. A few monoclonal antibodies are now available which bind selectively or exclusively to eccrine glands. 52 ,53 Such antibodies potentially may be used diagnostically to classify, on the basis of the glandular structure of origin, several of the more difficult benign and malignant append ageal tumors. Monoclonal Antibodies and Infectious Diseases 54 ,55 Monoclonal antibodies are now available against a wide variety of viral, bacterial, and fungal antigens. Although infectious diseases of the skin can be usually diagnosed on clinical grounds or by routine histologic examination and/or culture, monoclonal antibodies may prove useful when intact organisms and/or characteristic features are absent. Furthermore, the association of preceding infection with a secondary eruption, ie, herpes simplex infection followed by the development of erythema multiforme, may in some cases be confirmed by demonstration of microbial antigens within lesional skin. 56

Monoclonal Antibodies and Tumors of the Skin Malignant Melanoma. Within the past five years, at least 50 dif- . ferent monoclonal antibodies have been reported with relative binding specificity for antigens present on human melanoma cells; several are now commercially available for diagnostic applications. 32 ,57,58 Both cell surface and cytoplasmic melanoma-associated antigens have been defined and partially characterized using these antibodies. Unfortunately very few of these monoclonal antibodies appear to bind exclusively to melanoma cells, thereby somewhat limiting their applicability for diagnosis and therapy. Although melanomaassociated monoclonal antibodies may cross-react with a variety of normal and abnormal tissues, they most commonly cross-react with other tumors of neural origin, ie, astrocytoma and neuroblastoma. Since the latter type of tumors rarely are found in the skin, in practical terms these monoclonal antibodies still may be useful diagnostically in the evaluation of selected skin biopsies. Of importance, however, since many of the melanoma-associated monoclonal antibodies also cross-react with nevus cells and/or normal melanocytes, these antibodies will not be useful in distinguishing very early melanoma from precursor lesions, such as dysplastic nevi. Despite the problem of crossreactivity of some of these monoclonal antibodies with normal tissues, determination of quantitative differences in the expression of melanoma-associated antigens in normal tissues and melanoma may still permit their use diagnostically. Specifically, since many of these melanomaassociated antigens are expressed in markedly greater amounts by melanoma cells than by normal tissues, it may be possible with selected monoclonal antibodies to determine the minimum dilution

THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

necessary to eliminate background staining of tissues other than melanoma. As previously discussed, melanoma-associated monoclonal antibodies may be of great diagnostic use in the evaluation of severely anaplastic tumors arising in the skin.59,60 In most cases the use of a panel of melanoma-associated monoclonal antibodies and monoclonal antibodies directed against keratin should allow one to distinguish between melanoma and anaplastic tumors of epithelial origin. Unfortunately, many anti-melanoma monoclonal antibodies react wi"th antigens expressed in small or minute amounts by only a minority of melanoma cells;61,62 therefore, suspicious specimens should be evaluated with several antimelanoma monoclonal antibodies before it is concluded that the tumor is not melanoma. Other Tumors. Epithelialderived tumors can usually be defined by demonstrating staining by one or more monoclonal antibodies to keratin andlor desmosomal antigens such as desmoplakin. However, as previously noted, one possible exception is renal cell carcinoma, a tumor which not infrequently metastasizes to skin. Since monoclonal antibodies to the other four types of intermediate filaments (vimentin, desmin, neurofilament, glial fibrillary acidic protein) are now commercially available, it should be now possible to differentiate among anaplastic carcinomas, myogenic sarcomas, non-myogenic sarcomas, and neural tumors by demonstrating antigenic recognition by monoclonal antibodies to one or more of these groups of intermediate filaments. For example, rhabdomyosarcoma, leiomyosarcoma, and leiomyoma express both desmin and vimentin but lack cytokeratins; unfortunately, it is impossible to further distinguish among these three tumors on the basis of staining by such monoclonal antibodies. 63 -65 147

Monoclonal Antibodies and Skin Biopsy

Fibrosarcoma expresses vimentin and infrequently co-expresses desmin. 65 Only vim en tin is expressed in liposarcoma, angiosarcoma, dermatofibrosarcoma protru berans, fibrous histiocytoma, myxoma, malignant schwannoma, and lymphoma.63.65 Use of anti-cytokeratin monoclonal antibodies on skin biopsies of suspected metastatic adenocarcinoma of the stomach may allow easier identification of tumor ("signet ring") cells. 66 Finally, both keratin and neurofilaments are detectable within lesions of cutaneous neuroendocrine carcinoma. 67 Recently a new monoclonal antibody was described that has specificity for an intracellular antigen present in liposarcoma and in sebaceous glands. 68 Since no cross-reactivity was noted with other sarcomas examined, this monoclonal antibody may have diagnostic application in the identification of poorly differentiated liposarcoma and/or tumors arising from sebaceous glands. Since endothelial cells contain Factor VIII, it may be possible to diagnose early lesions of Kaposi's sarcoma using anti-Factor VIII monoclonal antibody. Similarly, Factor VIII should also be theoretically present within angiosarcoma. Both tumors are also detectable with a new monoclonal antibody reportedly specific for endothelial cells. 69 Extramammary Paget's disease and metastatic adenocarcinoma to the skin also may be identifiable using polyclonal or monoclonal antibodies to carcinoembryonic antigen. 51 Monoclonal Antibodies and Dermal Inflammatory

Infiltrates7o -78

Most non-tumorous skin eruptions are characterized by the presence of dermal inflammatory infiltrates composed of varying proportions of lymphocytes, histiocytes, eosinophils, polymorphonuclear leukocytes, plasma 148

cells, and/or mast cells. Determination of the pattern and composition of the infiltrate, as well as the nature of epidermal, dermal, and/or subcutaneous alterations, if present, usually results in correct diagnosis. . Over the past few years, a number of investigators have attempted to utilize commercially available monoclonal antibodies to specific lymphocyte subsets, particularly the OKT and Leu series, in order to better define and classify certain skin diseases, most notably cutaneous T-cell lymphoma (mycosis fungoides).70-75 Detailed discussion of lymphocyte-related monoclonal antibodies is beyond the scope of this review; therefore discussion will be limited to the application of monoclonal antibodies to the identification of cutaneous T-cell lymphoma. Unfortunately most of these studies have been performed on single skin biopsy specimens obtained from small numbers of patients with this as well as several unrelated disorders with the common finding of lymphohistiocytic dermal infiltrates. Since it is possible that the composition of lymphocytic infiltrates so detected may vary considerably dependent upon the time at which the biopsy was obtained, it is difficult to realistically make significant interpretations of the results of many of these studies, particularly as regards to diagnosis. In the case of cutaneous T-cell lymphoma, however, some statements can be made. For example, using such monoclonal antibodies no single pattern of reactivity has been defined that reliably distinguishes cutaneous T-cell lymphoma from other unrelated benign dermatoses. 74 Similarly, determination of OKT 4:0KT 8 ratios on peripheral blood lymphocytes has not proven helpful. For example, elevated ratios (6:1) have been reported not only in cutaneous T-cell lymphoma but also in at least one benign dermatosis, pityriasis rubra pilaris. 77 Despite

all of the above disclaimers, however, in a minority of patients with cutaneous T-cell lymphoma useful findings may include the demonstration of differential loss of surface antigens by T-cells, expression of immature lymphocyte phenotypes, and detection of lymphocytes reactive with monoclonal antibodies to OKT 10, OKT 9, and 5E9 antigens. 74 Recently two potentially useful monoclonal antibodies, BEl and BE2, have been described. 73 Both of these murine monoclonal antibodies were produced following immunization of experimental mice with leukemic helper T-cells from a patient with cutaneous Tcell lymphoma. Both antibodies bind to cell surfaces of a subset of lymphocytes (either circulating or present within affected skin and lymph nodes) from patients with cutaneous T-cell lymphoma. In contrast, neither shows increased reactivity with normal peripheral mononuclear cells (including normal helper cells) or a variety of normal human tissues . Both monoclonal antibodies also react with Epstein-Barr virus-transformed B-cell lines and selected longterm T-cell lines. BE2 also reacts with a subpopulation of lymphocytes from some patients having chronic lymphocytic leukemia. Immunoprecipitation studies suggest that different cellular antigens are recognized by each of these two monoclonal antibodies. To date, both of these monoclonal antibodies have been shown not to bind to skin biopsies from normal individuals or patients with several benign dermatoses, suggesting their possible utility in the diagnosis of cutaneous T-cell lymphoma. Of interest, peripheral blood lymphocytes were also positive in 16 of 21 patients with cutaneous T-cell lymphoma as yet unproven to have extracutaneous involvement 73 ; further studies will be necessary to determine the clinical implications of these findings. More recently, BE2 staining was also noted in skin biopsy specOctober 1985 Volume 290 Number 4

Fine

imens from one patient with lymphomatoid papulosis, one patient with cutaneous B-cell lymphoma, and on peripheral blood lymphocytes of 43% of 37 patients with AIDS.75,78 Considering these latter findings, some caution should be used therefore in the interpretation of positive staining with the BE2 monoclonal antibody since it is now clear that crossreactivity may occur with lymphocytes from patients with some other conditions. Limitations in the Use of Monoclonal Antibodies to Assess Skin Biopsies

As discussed earlier, great care must be taken to define the extent of cross-reactivity of a particular monoclonal antibody prior to its use for diagnostic purposes. This is particularly the case with monoclonal antibodies to tumor antigens, since the latter may be also expressed in small amounts in some normal tissues as well as by unrelated tumors. Furthermore, many antigens recognized by monoclonal antibodies are rather labile; as such, they may be antigenically altered, destroyed, or lost during the process of routine tissue fixation. Although still usable on unfixed fresh-frozen tissues, ie, antibodies utilized in immunofluorescence mapping studies, the need for monoclonal antibody studies usually becomes apparent after examination of fixed tissue proves to be non-diagnostic by routine histologic parameters. Therefore, in general, monoclonal antibodies of most practical diagnostic usefulness will be those which recognize tissue antigens unaltered by formalin fixation and routine tissue processing. Summary

Within the past few years, application of monoclonal antibody technology to dermatologic research has yielded considerable new insight into the biochemical and ultrastructural composition of

human skin. More recently, these same antibodies have been shown to have practical applications in the diagnosis of both benign and malignant conditions involving the skin. It is likely in the near future that the use of these immunohistochemical reagents will become a routine part of the evaluation of selected skin biopsies as more of the better characterized monoclonal antibodies become commercially available. References 1. Diamond BA, Yelton DE, Scharff MD: Monoclonal antibodies: A new technology for producing serologic reagents. N Engl J Med 304: 1344-1349, 1981. 2. Oi VT, Herzenberg LA: Immunoglobulin-producing hybrid cell lines, in Mishe\1 BB, Shiju SM (eds): Selected Methods in Cellular Immunology. San Francisco, WH Freeman, 1980, pp 351-372. 3. Galfre G, Milstein C: Preparation of monoclonal antibodies: Strategies and procedures. Methods Enzymol 73:3-46, 1981. 4. Osborn M, Weber K: Biology of disease. Tumor diagnosis by intermediate filament typing: A novel tool for surgical pathology. Lab Invest 48:372-394, 1983. 5. Woodcock-Mitchell J, Eichner R, Nelson WG, Sun T-T: Immunolocalization of keratin properties in human epidermis using monoclonal antibodies. J Cell Bioi 95:580-588, 1982. 6. Eichner R, Bonitz P, Sun T-T: Keratin families: Identification of constant and variable members using monoclonal antibodies. J Invest Dermatol 80:315, 1983. 7. Sun T-T, Eichner R, Nelson WG, Tseng SCG, Weiss RA, Jarvinen M, Woodcock-Mitchell J: Keratin classes: Molecular markers for different types of epithelial differentiation. J Invest Dermatol 81:109-115, 1983. 8. Weiss RA, Sun T-T: A 48 K and 56 K keratin as molecular markers of epidermal hyperproliferation : Monoclonal antibody analysis. J Invest Dermatol 80:337, 1983. 9. Weiss RA, Guillet GYA, Freedberg 1M, Farmer ER, Sma\1 EA, Weiss MM, Sun T-T: The use of monoclonal antibody to keratin in human epidermal disease: Alterations in immunohistochemical staining pattern. J Invest Dermatol 81:224-230,1983. 10. Chardonnet Y, Beauve P, Viae J, Schmitt D: Warts: Langerhans ce\1s and T lymphocytes in local immune response. J Invest Dermatol 80:348 , 1983.

THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

11. Oseroff AR, Roth R, Lipman S, Morhenn VB: Use of a murine monoclonal antibody which binds to malignant keratinocytes to detect tumor ce\1s in microscopically controlled surgery. J Am Acad Dermatol 8:616-619 , 1983. 12. Gatter KC, Alcock C, Heryet A, Pulford KA, Heyderman E, TaylorPapadimitriou J, Stein H, Mason DY: The differential diagnosis of routinely processed anaplastic tumors using monoclonal antibodies. Am J Clin Pathol 82:33-43, 1984. 13. Nagle RB, McDaniel KM, Clark VA, Payne CM: The use of anti keratin antibodies in the diagnosis of human neoplasms. Am J Clin Pathol 79:458-466, 1983. 14. Moll R, Franke WW, Schiller DL, Geiger S , Krepler R: The catalogue of human cytokeratin polypeptides: Patterns of expression of cytokeratins in normal epithelia, tumors and cultured cells. Cell 31 :11-24, 1982. 15. Murphy GF: Cytokeratin typing of cutaneous tumors: A new immunochemical probe for cellular differentiation and malignant transformation. J Invest Dermatol84:1-2, 1985. 16. Moll I, Moll R: Cells of extramammary Paget's disease express cytokeratins from those of epidermal cells. J Invest Dermatol 84:3-8, 1985. 17. Hintner H, Fine JD, Neises GR, Steinart P, Lawley TJ: Monoclonal antibody reacting with "profilaggrin." Clinical Research 31:264A, 1983. 18. Sybert VP, Dale BA, Holbrook KA: Ichthyosis vulgaris: Identification of a defect in synthesis of filaggrin correlated with an absence of keratohyaline granules . J Invest Dermatol 84: 191-194, 1985. 19. Stanley JR, Koulu L, Klaus-Kovtun V, Steinberg MS: A monoclonal antibody against desmoglein I (DGI) binds to pemphigus foliaceus (PF) antigen . Clinical Research 33:684A, 1985. 20. Fine JD, Breathnach SM, Fox PA, Neises GR, Stanley JR, Katz SI: Monoclonal antibodies in dermatologic research: Studies with a unique keratinocyte-specific cell-surface antigen defined by a monoclonal antibody (KF-2). Am J Dermatopathol (in press). 21. Eto H, Tazawa T, Ito M, Matsumoto M, Hashimoto K: Differential staining of acantholytic cells in pemphigus vulgaris, Darier's disease and HaileyHailey disease with monoclonal antidesmosome antibody HKl. Clinical Research 33:636A, 1985. 22. Franke WW, Moll R, Mueller H, Schmid E, Kuhn C, Krepler R, Artlieb U, DenkH: Immunocytochemical identification of epithelium-derived human tumors with antibodies to desmosomal plaque proteins. Proc Natl Acad Sci USA 80:543-547, 1983. 23. Mansbridge J, Knapp A, Strefling A: Evidence for an alternative pathway of

149

Monoclonal Antibodies and Skin Biopsy

keratinocyte maturation in psoriasis from an antigen found in psoriatic but not normal epidermis. J Invest Dermatol 83:296-301, 1984. 24. Strefling AM, Knapp AM, Mansbridge IN: Histologic distribution of staining by a monoclonal antibody (~1-3) in psoriasis and occurrence of~I-3 antigen in other cutaneous diseases. J Invest Dermatol 84:100-104, 1985. 25. Rowden G, Lewis MG, Sullivan AK: Ia antigen expression on human epidermal Langerhans cells. Nature 268:247-248, 1977. 26. Fithian E, Kung P, Goldstein G, Rubenfeld M, Fenoglio C, Edelson R: Reactivity of Langerhans' cells with hybridoma antibody. Proc Natl Acad Sci USA 78:2541-2544, 1981. 27. Kashihara M, Ueda M, Horiguchi Y, Furukawa F, Hanaoka M, Imamura S: A monoclonal antibody specifically reactive to human Langerhans cell. Clinical Research 33:653A, 1985. 28. Belsito DV, Sanchez MR, Baer RL, Valentine F, Thorbecke GJ: Reduced Langerhans' cell Ia antigen and ATPase activity in patients with the acquired immunodeficiency syndrome. N Engl J Med 310:1279-1282, 1984. 29. Saurat JH, Chavaz P, Carraux P, Didierjean L: A human monoclonal antibody reacting with Merkel cells: Immunofluorescence, immunoperoxidase, and immunoelectron microscopy. J Invest Dermatol 81:249-253, 1983. 30. Saurat J-H, Didierjean L, Skalli 0, Siegenthaler G, Gabbiani G: The intermediate filament protein of rabbit normal epidermal Merkel cells are cytokeratins. J Invest Dermatol 83:431-435, 1984. 31. Tomita Y, Montague PM, Hearing VJ: Monoclonal antibodies to mouse T4tyrosinase identify human melanocytes. Clinical Research 33:690A, 1985. 32. Fine JD: Current and potential applications of monoclonal antibodies in the study, diagnosis, and treatment of malignant melanoma and non-lymphocytic tumors of the skin, in Cutaneous Surgery-1985. New York, ThiemeStratton, to be published. 33. De Waal RMW, Semeijn JT, Cornelissen IMH, Ramaekers FCS: Epidermal Langerhans cells contain intermediate-sized filaments of the vimentin type: An immunocytologic study. J Invest Dermatol 82:602-604, 1984. 34. Foidart JM, Bere EW, Yaar M, Rennard SI, Gullino M, Martin GR, Katz SI: Distribution and immunoelectron microscopic localization of laminin, a non-collagenous basement membrane glycoprotein. Lab Invest 42:336-342, 1980. 35. Yaoita H, Foidart JM, Katz SI: Localization of the collagenous component in skin basement membranes. J

150

Invest Dermatol 70:191-193, 1978. 36. Paller AS, Queen LL, Woodley DT, Gammon WR, O'Keefe EJ, Briggaman RA: A mouse monoclonal antibody against a newly discovered basement membrane component, the epidermolysis bullosa acquisita antigen. J Invest Dermatol 84:215-217, 1985. 37. Fine JD: Epidermolysis bullosa: Variability of expression of cicatricial pemphigoid, bullous pemphigoid, and epidermolysis bullosa acquisita antigens. J Invest Dermatol, to be published. 38. Breathnach SM, Fox PA, Neises GR, Stanley JR, Katz SI: A unique squamous epithelial basement membrane antigen defined by a monoclonal antibody (KF-l). J Invest Dermatol 80:392-395, 1983. 39. Bernard BA: Characterization of KF-1 monoclonal antibody. Clinical Research 33:625A, 1985. 40. Fine JD, Breathnach SM, Hintner H, Katz SI: KF-1 monoclonal antibody defines a specific basement membrane antigen defect in dystrophic forms of epidermolysis bullosa. J Invest Dermatol 82:35-38, 1984. 41. Fine JD, Smith LT, Holbrook KA, Katz SI: The appearance of four basement membrane zone antigens in developing human fetal skin. J Invest Dermatol 83:66-69, 1984. 42. Goldsmith LA, Briggaman RA: Monoclonal antibodies to anchoring fibrils for the diagnosis of epidermolysis bullosa. J Invest Dermatol 81:464-466, 1983. 43. Lane AT, Helm KF, Goldsmith LA: Identification of bullous pemphigoid, pemphigus, laminin, and anchoring fibril antigens in human fetal skin. J Invest Dermatol 84:27-30,1985. 44. Hintner H, Stingl G, Schuler G, Fritsch P, Stanley J, Katz, Wolff K: Immunofluorescence mapping of antigenic determinants within the dermalepidermal junction in mechanobullous diseases. J Invest Dermatol 68: 119-124, 1981. 45. Weber L, Krieg T, Muller PK, Kirsch E, Tompl R: Immunofluorescent localization of type IV collagen and laminin in human skin and its application injunctional zone pathology. Br J Dermatol 106:267-273, 1982. 46. Pearson RW: Epidermolysis bullosa, porphyria cutanea tarda, and erythema multiforme, in Zelickson AS (ed): Ultrastructure of Normal and Abnormal Skin. Philadelphia, Lea & Febiger, 1967, pp 320-329. 47. Bauer EA, Briggaman RA: The mechanobullous diseases (epidermolysis bullosa), in Fitzpatrick TB, Eisen AZ, Wolff K, Freedberg 1M, Austen KF (eds): Dermatology in General Medicine, ed 2. New York, McGraw-Hill Book Co., 1979, pp 334-347. 48. Miller EJ, Gay S: The multiple types

and forms of collagen. Methods Enzymol 82A:3-32, 1982. 49. Gay S: The immunology of collagen, in Wagner BM, Fleishmajer R, Kaufman N (eds): Monographs in Pathology: Connective Tissue Diseases. Baltimore, Williams & Wilkins, 1983, pp 120-128. 50. Osborn M: Intermediate filaments as histologic markers: an overview. J Invest Dermatol 81:104s-107s, 1983. 51. Pennys NS: Immunoperoxidase applications in skin pathology, in Callen JP (ed): Current Issues in Dermatology, Vol. 1, Boston, GK Hall Medical Publishers, 1984, pp 173-194. 52. Eto H, Matsumoto M, Kobayashi H, Mehregan A, Hashimoto K: Eccrine gland associated antigens. A demonstration by monoclonal antibodies. J Invest Dermatol 80:339, 1983. 53. Kanitakis J, Schmitt D, Bernard A, Boumsell L, Thivolet J: Anti-D47: A monoclonal antibody reacting with the secretary cells of human eccrine sweat glands. Br J Dermatol 109:509-513, 1983. 54. Nowinski RC, Tam MR, Goldstein LC, Stong L, Kuo C-C, Corey L, Stamm WE, Handsfield HH, Knapp JS, Holmes KK: Monoclonal antibodies for diagnosis of infectious diseases in humans. Science 219:637-644, 1983. 55. Engleberg NC, Eisenstein BI: The impact of new cloning techniques on the diagnosis and treatment of infectious diseases. N Engl J Med 311:892-901, 1984. 56. Orton PW, Huff JC, Tonnesen MG, Weston WL: Detection of a herpes simplex viral antigen in skin lesions of erythema multiforme. Ann Intern Med 101:48-50, 1984. 57. Imai K, Wilson BS, Kay NE, Ferrone S: Monoclonal antibodies to human melanoma cells: Comparison of serological results of several laboratories and molecular profile of melanoma-associated antigens, in Hammerling GJ, Hammerling U, Kearney JF (eds): Monoclonal Antibodies and T-Cell Hybridomas: Perspectives and Technical Advances. New York, Elsevier/North Holland Biomedical Press, 1981, pp 183-190. 58. Houghton AN: Identification of differentiation antigens of melanoma and melanocytes by mouse and human monoclonal antibodies. Transplant Proc 16:351-354, 1984. 59. Van Duinen SG, Ruiter DJ, Hageman P, Vennegoor C, Dickersin GR, Scheffer E, Rumke P: Immunohistochemical and histochemical tools in the diagnosis of amelanotic melanoma. Cancer 53:1566-1573, 1984. 60. Gatter KC, Pulford KA, Vanstapel MJ, Roach B, Mortimer P, Woolston RE, Taylor-Papadimitriou J, Lane EB, Mason DY: An immunohistological study of benign and malignant skin tumors: epithelial aspects. HistoOctober 1985 Volume 290 Number 4

Fine

pathology 8:209-227, 1984. 61. Libert A, Ghanem G, Arnould R, Vercammen-Grandjean A, Carrel S, Lejeune F: Monoclonal antibodies to human melanoma-associated antigens: Study of their specificity and visualization at the cellular level of the antigenic distribution. Anticancer Research 3:219-225, 1983. 62. Natali PG, Viora M, Nicotra MR, Giacomini P, Bigotti A, Ferrone S: Antigenic heterogeneity of skin tumors of non melanocyte origin: Analysis with monoclonal antibodies to tumor-associated antigens and to histocompatibility antigens. JNCI 71:439-447,1983. 63. Altmannsberger M, Osborn M, Treuner J, Holscher A, Weber K, Schauer A: Diagnosis of human childhood rhabdomyosarcoma by antibodies to desmin the structural protein of muscle specific intermediate filaments. Virchows Arch (Cell Pathol) 39:203-215, 1982. 64. Gabbiani G, Kapanci Y, Barazzone P, Franke WW: Immunochemical identification of intermediate-sized filaments in human neoplastic cells. Am J Pathol104:206-216, 1981. 65. Miettinen M, Lehto V-P, Badley RA, Virtanen I: Expression of intermediate filaments in soft tissue sarcomas. Int J Cancer 30:541-546,1982. 66. Altmannsberger M, Weber K, Holscher

A, Schauer A, Osborn M: Antibodies to intermediate filaments as diagnostic tools: Human gastrointestinal carcinomas express keratin. Lab Invest 46:520-526, 1982. 67. Hoefler H, Rauch H-J, Kerl H, Denk H: New immunocytochemical observations with diagnostic significance in cutaneous neuroendocrine carcinoma. Am J Dermatopathol 6:525-530, 1984. 68. Matsuo S, Penneys N, ZiegelsWeissman J, Adachi K: A new monoclonal antibody which identifies liposarcoma (LS) cells. Clinical Research 33(Abstract):665, 1985. 69. Matsuo S, Penneys N, Nadji M, SeigelsWeissman J, Adachi K: A new monoclonal antibody which identifies endothelial cells (EC). Clinical Research 33(Abstract):665, 1985. 70. Chu AC: Monoclonal antibodies in dermatology. J Royal Soc Med 76:1-4, 1983. 71. Kung PC, Berger CL, Estabrook A, Edelson RL: Monoclonal antibodies for clinical investigation of human T lymphocytes. Int J Dermatol 22:67-74, 1983. 72. McMillan EM: Monoclonal antibodies and cutaneous T cell lymphoma. Theoretical and practical considerations. J Am Acad Dermatol 12:102-114, 1985. 73. Berger CL, Morrison S, Chu A, Patterson J, Estabrook A, Takezaki S, Sharon J, Warburton D, Irigoyan 0, Edelson

THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

74.

75.

76.

77.

78.

RL: Diagnosis of cutaneous T celllymphoma by use of monoclonal antibodies reactive with tumor-associated antigens. J ClinInvest 70:1205-1215,1982. Chu AC: The use of monoclonal antibodies in the in situ identification ofTcell subpopulations in cutaneous T-~ell lymphoma. J Cutan Pathol10:4 79-498, 1983. Prendeville J, Smith NP, Berger CL, Edelson R, Chu AC: Monoclonal antibodies in the tissue diagnosis of cutaneous T cell lymphoma. J Invest Dermatol 82:560, 1984. Berger CL, Edelson RL: Peripheral blood of patients with cutaneous T-cell lymphoma: Studies using monoclonal antibodies. J Cutan Pathol1 0:467 -4 78, 1983. Chu AC, Berger CL, Edelson RL, Spittle MF, Smith NP: Cutaneous T cell lymphoma-diagnosis using monoclonal antibodies against normal and malignant T cells. J Invest Dermatol 80:333, 1983. Berger CL, Rehle T, Friedman-Kien AE, DiFranco M, Ostreicher R, Knobler R, Laubenstein LJ, Edelson R: Common membrane antigen on lymphocytes from cutaneous T cell lymphoma and acquired immune deficiency syndrome patients. J Invest Dermatol 82:409, 1984.

151