Immunohistochemical Evaluation of Vascular Neoplasms PAUL MARK
E. SWANSON, R. WICK,
MD
MD
he histologic features of vascular neoplasms arising in the superficial soft tissue and skin are considerably more complex than one might predict from the appearance of the normal endothelial cell. This attribute of endothelial proliferations may cause substantial diagnostic confusion in some instances. The most troublesome to the pathologist are those neoplasms in which a spindled or an epithelioid population predominates. Because spindle cell tumors of the skin are addressed elsewhere in this monograph, much of the ensuing discussion will focus on overtly vasogenic neoplasms and their epithelioid histologic variants. The purpose of this presentation is not to deal with these entities in a comprehensive manner, because their morphologic features have been discussed at length elsewhere.1-20 Instead, we will review the application of immunohistochemical techniques to the routine evaluation of vascular proliferations. The fundamentals of immunohistochemistry are no longer foreign concepts to most practitioners of dermatopathology, nor is there much disagreement as to the relative efficacy of the various methodologies that are available. These include the peroxidase anti-peroxidase (PAP)** and the avidin-biotin-peroxidase complex (ABC) techniques** and variations or modifications thereof.23 In most settings, we prefer the latter procedure, although most of the antibodies applicable to the evaluation of endothelial differentiation are not dependant on methodologic choices. The greatest advantage of the ABC technique is that its relative efficiency (compared to PAP) has obviated the need for certain modifications designed to enhance the sensitivity of the assay. Various aldehyde
T
From the Department of Pathology, Washington University School of Medicine, St. Louis, Missouri. Address correspondence to: Paul E. Swanson, M.D., Division of Anatomic Pathology, Barnes Hospital, 2 Barnes Hospital Plaza, St. Louis MO
63110.
0 2991 by Elsevier Science Publishing
Co., Inc.
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and alcohol-based fixatives have been employed to improve the preservation of endothelial antigens,24*25 but in many instances, routinely processed tissues yield readily interpretable immunohistochemical results. Similarly, the use of protease digestion, to “unmask’ fixation-altered cellular antigens, was historically considered a requirement in the immunohistochemical detection of some endothelial determinants.25-27 With current commercial ABC products and antibody reagents, however, this manipulation has become unnecessary in most cases. Various cell products have been described (in addition to vWF), which, when detected immunohistochemically, point towards endothelial differentiation. The conjoint use of these markers often simplifies the diagnostic problems posed by vascular neoplasms, a concept that is amplified in the ensuing discussion and summarized in Tables 1 and 2.
Factor VIII-Related Antigen and Thrombomodulin Despite the unique functions performed by endothelial cells, relatively few biological markers consistently label neoplastic endothelium. The emphasis on the term neoplastic is important, because several cell products elaborated by normal or reactive endothelium are underexpressed, or, for practical purposes, are absent in a large proportion of benign and malignant vascular tumors. Perhaps the best known of these substances is von Willebrands factor (vWF : Factor VIII-related antigen). This clotting co-factor is actively synthesized by virtually all non-lymphatic endothelial cells,2**29 and is probably stored in and transported by the Weibel-Palade bodies that ultrastructurally characterize such cells.30 Commercially available monoclonal and polyclonal antibodies with proven specificity for vWF have been successfully applied to the immunohistochemical analysis of routinely processed tissues. Nonetheless, the diagnostic value of
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2991;9:243-253 Table 1. Immunohistochemisty
in the Differential Diagnosis of Hemanxioma
vWF
Tumor Hemangioma Lymphangioma Glomus Tumor Hemangiopericytoma Angiomatoid Fibrous Histiocytoma Cellular Angiolipoma Spitz Nevus Spiradenoma
UEA
TM
+/0 0 0 0
+ + 0 0 0
+ 0 0
+ 0 0
mACT
CK
EMA
HMB45
SIOO
+ + +/+ 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
+ 0 0
0 0 0
0 0 +
0 0 +
0 + 0
+ + +
C4L
ACT
+ 0 0 0 0
+ 0 + +/0
0 nt nt
+/0 0
Abbreviations: vWF= von Willebrand’s factor (Fnctor VIII-related antigen); UEA = Ulex europaeus agglutinin I; TM = thrombomodulin; C4L = collagen type ZV/laminin; ACT = actin; mACT = muscle-specific actin; CK = cytokeratin; EMA = epifhelial membrane antigen; nt = not tested.
these antibodies in the recognition of vascular neoplasia is limited.31 Indeed, even when protease digestion and other enhancement techniques are applied, only half of benign vascular lesions are reproducibly labeled for vWF, and fewer than 25% of malignant endothelial lesions (Figure 1) are similarly immunoreactive.26~27~31-37 Nonetheless, antibodies to vWF are a specific marker of endothelial differentiation, and accordingly remain an important part of the immunohistochemical diagnosis of vascular tumors. Expectedly, neoplasms that contain predominantly non-endothelial vasofonnative elements, including glomus tumor and hemangiopericytoma, are typically negative for vWF.25,38,39 Somewhat better results have been obtained using antibodies to the endothelial-selective product thrombomodulin (TM).40,41 This substance, which is essentially an antagonist of vWF, acts by forming a stable stoichiometric complex with thrombin, which, in turn, manifests enhanced activation of the anticoagulant protein C. In contrast to vWF, TM is more strongly expressed in malignant endothelial proliferations. In one published series, polyclonal antibodies to TM labeled each of 14 angiosarco-
Table 2. lmmunohistochemisty
vWF
Tumor Epithelioid
Angiosarcoma
in the Differential
and
mas, as well as examples of epithelioid hemangioendothelioma, and various benign hemangiomas.41 Notably, neither the glomus cells nor the endothelial cells of glomus tumor were convincingly stained. The reciprocal relationship between the expression of vWF and TM in vascular tumors has not been well-explained, although the impact it may have on the absence of effective hemostasis in malignant endothelial lesions has been the subject of some conjecture. 41 The greatest weakness of antibodies to this marker is that TM-reactive substances are also elaborated by certain non-endothelial neoplasms, including squamous cell carcinoma41 and trophoblastic neoplasms. 42 The former is an important concern, because of the close histologic similarities that may exist between epithelioid angiosarcoma and the so-called acantholytic variant of squamous cell carcinoma.43 Further studies of endothelial and non-vascular neoplasms may help to define the role of this marker. Unfortunately, antibodies to TM are not yet commercially available. Antibodies have been raised to a family of endothelial products called endothelins. I4 However, these vasoactive oligopeptides are not selectively expressed by endothe-
Diagnosis of Epithelioid
TM
UEA
Endothelial
DES/
Neoplasms
CV
mACT
EMA
HMB
SIOO
LCA
DR
C4L
+/-
+
+
0
0
0
0
0
0
+
Hemangioendothelioma Epithelioid Sarcoma Epithelioid Leiomyosarcoma Malignant Lymphoma (including “malignant angioendotheliomatosis”)
0 0 0
nt 0 0
+/0 0
0 + 0
+ 0t 0
0 0 0
0’ 0t 0
0 0 +
+/+/+
0 + 0
Metastatic Carcinoma Adenoid Squamous Cell Carcinoma Malignant Melanoma
0 0 0
nt +/0
+/0 0
0 0 0
+ + 0
0 0 +
+/0 +
0 0 0
+/0 +
+/0 0
Abbreviations: uWF = van Willebrand’sfuctor; TM = thrombomodulin; UEA = UIex europaeus agglufinin I; DES = desmin; mACT = muscle-specific actin; CK = cytokerafin; EMA = epithelial membrane antigen; LCA = leukocyte common antigen; DR = P&l-DR antigen; C4L = collagen type IV/Zaminin; nt = not tested. l Occasional cases nre SIOO-positive. f Occasional cutaneous leiomyosnrcomas may be reactive for CK, EMA, and SIOO.
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Figure 1. von Willebrand Factor (vWF: Factor VIII-related Antigen) in Angiosarcoma. lmmunoreactivity for vWF is localized to endothelial cytoplasm in fewer than 25% of angiosarcomas.
lium, and are thus of little value as immunohistochemical markers.45-49 Similar comments apply to the detection of other endothelial-associated products, such as 5’-nucleotidase, 6-keto-prostaglandin-F1-alpha,49 antithrombin 111, thrombospondm-and angiotensin converting enzyme.50
Monoclonal Antibodies to Other EndotheliumSelective Determinants Considerable effort has been expended in the production of monoclonal antibodies to intact and cultured endothelial cells or cell lysates. Of these, the antibodies PAL-E51,* EN-4,24 and BW20052,53 have received the most attention. The greatest drawback to the routine application of certain of these antibodies is their requirement for frozen or *Actually, PAL-E was one of several antibody clones produced by a mouse thaf had been immunized with cells recovered from a lymph nodal metasfasis of malignant melanoma. 51 We can only assume that endothelial cells contaminating the preparation were also injected.
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specially prepared tissue substrates. Among the latter reagents, only BW200 exhibits reliable reactivity in formalin-fixed, paraffin-embedded tissues. Nonetheless, given the availability of appropriate tissues, these antibodies are of potential value for two reasons. One is their putative specificity for endothelial cells. The second is the selective non-reactivity with endothelial lymphatics exhibited by PAL-E.51 The application of a panel of these markers, together with antibodies to vWF and TM, may conceivably distinguish between lesions of vascular or lymphatic endothelial origin. The practical use of this information is limited, however, and is likely to be of little consequence to the clinical diagnosis and treatment of angiosarcoma. The same concerns may be voiced regarding the applicability of the antibody HECA-452, a monoclonal product with putative specificity for cells differentiating as postcapillary “high endothelium.” 54 These reagents are of greater interest to those exploring the differentiation and function of normal endothelium, and its interaction with lymphoid and extravascular tissues.55 Several other antibodies with selectivity for endothelium have been described, including E92,56,57 HC-1,58 0KM-1,59 and 0KM-5.59 In each instance, however, significant reactivity also has been noted with hematolymphoid or non-endothelial mesenchymal cell populations, setting the stage for the inadvertant misinterpretation of an intra- or perivascular lymphoid proliferation as endothelial in nature. Tumors with affinity for lymphoid elements, such as the endovascular papillary angioendothelioma,3*60*61or others composed of malignant lymphoid cells, such as intravascular lymphomatosis (“malignant angioendotheliomatosis”)62-66 are perhaps the more important examples of this potential interpretive pitfall. In each case, antibodies directed at lymphoid-specific determinants, such as leucocyte common antigen, or B- and T-lineage selective antigens,67,68 will correctly identify the lymphoid elements in such lesions.
Blood Group-Related Antigens The expression of type-specific ABO blood group antigens is a normal function of non-lymphatic endothelium. In neoplastic alterations, host type-specificity may be lost, but ABO substances persist in most instances.69-73 It follows from this observation that antibodies to A, B, and H antigen may be of value in the detection of endothelial differentiation. Specifically, antibodies to H-substance have been shown to label the majority of benign and malignant endothelial proliferations.69,74 Unfortunately, as with other markers discussed above, the presentation of ABO antigens is not a unique function of endothelium among non-hematopoietic tissues. Normal and neoplas-
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Figure 2. Ulex europaeus 1 agglutinin (LIE& in Epithelioid angiosarcoma. More commonly identified than vWF, UF.A crisply labels endothelial cell surfaces.
tic epidermal and cutaneous adnexal epithelium, as well as some non-vascular sarcomas, may also produce immunodetectable H antigen.25~69~70~72~73 These comments are of considerable importance to our understanding of the utility of the plant lectin Ulex europaeus I agglutinin (UEA) in the histochemical evaluation of vascular tumors.75 Although the ability of UEA to recognize and bind to terminal fucosyl residues allows it to label a variety of glycoproteinaceous substances, the most common of the latter in human tissues is the H antigen. As a result, this lectin, in a manner similar to that of antibodies to H substance, is capable of recognizing a variety of non-vascular neoplasms, including certain adenocarcinomas and epithelioid sarcoma, each of which may be occasionally considered in the differential diagnosis of epithelioid vascular lesions.69,70,72*73Nonetheless, the striking sensitivity of this marker for endothelial malignancies (greater than 90%)76-78 proves its value as a marker for vascular differentiation (Figure 2) when an immunohistochemical analysis otherwise excludes other diagnostic alternatives. Together with antibodies to vWF, this lectin is useful in the immunohistochemical evaluation of cutaneous and soft tissue neoplasms. Antibodies to human histocompatibility antigens, including HLA-A,B,C, and beta-2-microglobulin (Class I) and HLA-DR (Class II), are of limited value in the diagnostic evaluation of endothelial tumors (and human neoplasia in general). Class I antigens are widely expressed by endothelium, mesenchyme and virtually all epithelial tissues, and induction of Class II antigens also may be
Clinics in Dermatology 1992:9:243-253 observed in a variety of reactive and neoplastic disorders of these tissues.7*,79,80 Nonetheless, the demonstration of HLA-DR in an epithelioid malignancy of skin may be of some practical use, as examples of angiosarcoma studied to date have not been immunoreactive for these determinants, whereas epithelioid sarcoma and malignant melanoma may be HLA-DR positive (Figure 3).*O Interestingly, Kaposi’s sarcoma may occasionally contain HLA-DR reactive cells, as well.sO The latter observation, taken together with the absence of reactivity for ABH antigens, and, in our experience, only sporadic or unreliable reactivity for vWF,*~ has provided suggestive immunohistochemical evidence that a fundamental difference exists between Kaposi’s sarcoma and other endothelial neoplasms as a group. There is, of course, considerable disagreement on this and at least one recent analysis of KS topic, 7*32,34-37*81-*3 purports to show both vWF and UEA staining in a majority of cases. ~4Nonetheless, the general lack of consistency in the staining characteristics of KS in other studies emphasizes the capricious nature of this neoplasm, and underscores our current inability to understand it fully using routinely available techniques. A similar problem has been encountered in the evaluation of the recently char“spindle-cell hemangioendothelioma” acterized (SCH).15J7 Although this tumor is clearly vasogenic and shows areas resembling cavernous hemangioma and epithelioid hemangioendothelioma, the ultrastructural and immunophenotypic characteristics of the spindled cells in SCH are not clearly endothelial in nature. The lack of vWF staining in the latter cells in the few examples studied to date,15 suggests that innately endothelial proliferations may have the capacity to evolve into cell populations with indeterminate or non-endothelial mesenchyma1 differentiation. Further study of Kaposi’s sarcoma and SCH may help to substantiate this hypothesis.
Matrical Proteins and Matrical Binding Proteins A consistent feature of normal vascular endothelium is its ability to elaborate an intact basement membrane; this function is at least partially preserved in neoplastic proliferations as well. Antibodies to the matrical components collagen type IV and laminin have been applied to the diagnosis of vascular neoplasms.71,85-*9 While there have been insufficient data presented with which to evaluate their overall sensitivity for endothelial lesions, it should be evident that they are non-specific. Even so, the expression of these substances by vascular neoplasms has been tested in two specific settings. First, it has been suggested that neither type IV collagen nor laminin is typically produced by lymphatic endothelium or its neoplasms, and like PAL-E, they may aid in the separation of
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Figure 3. Class I1 Histocompatibility Antigens in Epithelioid Malignancies. Epithelioid angiosarcoma does not express HLADR (left), whereas histologically similar examples of malignant lymphatic from vascular lesions in skin and other sites (Figure 4).a’ However, the recent demonstration of laminin reactivity in both capillaries and lymphatics in gastric tissue calls this general assumption into question.90 Second, several authors have suggested that matrical proteins are present to a quantifiably lesser degree in angiosarcoma and Kaposi’s sarcoma, compared to benign vascular neoplasms. ‘~JX Whereas this postulate may have biochemical and morphologic support, there is no compelling evidence that immunohistochemistry can reliably detect useful differences in this regard. Attempts to establish the malignancy of a neoplasm on the basis of immunohistochemical staining are a tenuous and unwise pursuit at best; the proposition that matrical proteins might serve such a purpose only promulgates a dangerous fallacy. Another approach to the identification of vascular tumors is the detection of matrical ligand-binding substances at the endothelial cell surface. Perhaps the best understood elements among cell adhesion proteins are the integrins, a complex but interrelated group of heterodimeric molecules with relative specificity for different
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melanoma may be reactive (right), using the monoclonal antibody LN3.
matrical peptides. 91 No one integrin is specific for endothelium, although the presence of the integr-in VIA-4 or similar proteins in part characterizes certain lymphoidassociated endothelial cell populations.92 For reasons described above, these proteins are of considerable interest to students of endothelial cell function, but they have little application to diagnostic immunohistochemistry.
Non-Endothelial Markers of Importance in the Diagnosis of Vascular Lesions As the foregoing discussion implies, the immunohistochemical recognition of endothelial tumors usually relies on information other than reactivity for endothelial-related antigens. Bearing in mind that the lack of such reactivity may in part be the result of fixation-induced loss or alteration of these determinants, any immunohistochemica1 analysis of a given neoplasm requires an assessment of the presence of a substance known to be expressed. Our recommendation along these lines is that antibodies to vimentin be applied, in order to evaluate staining of mesenchymal elements. One of the latter, of
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Figure 4. Matrical Proteins in Benign Vascular Tumors. Antibodies to laminin stain the basement membrane around
course, is endothelium, which contains vimentin as its principal structural protein. Not surprisingly, virtually all vascular neoplasms, unless handled improperly during processing, are vimentin-reactive (Figure 5).25*93-95 Other intermediate filaments of interest in the evaluation of endothelial tumors are desmin and cytokeratin, not because they are normally expressed by endothelium, but because their detection generally excludes the diagnosis of endothelial differentiation. The former of these moieties is characteristic of normal and neoplastic myogenie elements,93-95 and together with muscle-specific actin isoforms, constitutes the basis for the detection of muscle differentiation in cutaneous neoplasms.25,96*97 Cytokeratins serve a similar function, as their presence typically equates with epithelial differentiation. Reactivity for these peptides in primary and metastatic carcinomas of the skin, and epithelioid sarcoma,24,25*93-95 remains the most convincing indication that the alternative diagnosis of an epithelioid vascular neoplasm is incorrect. Recent reports, however, have questioned this conclusion. In four separate studies, immunoreactivity for low-molecular weight cytokeratins has been described in angiosarcoma and epithelioid hemangioendothelioma.98-10* At
neoplastic endothelial cells in cavernous hemangioma usually do not stain lymphangiomas (right).
(left), but
this point, biochemical demonstration of cytokeratins in these tissues is lacking. Moreover, evaluation of the methodologic aspects of such studies suggests that the cytokeratin “positivity” they report is unreliable, in that it was obtained only at high antibody concentration. In light of these claims, a more extended panel of antibodies to epithelial determinants may provide some solace to the observer who is unwilling to rely on cytokeratins in this setting. Epithelial membrane antigen (EMA) and carcinoembryonic antigen, among others, represent determinants that are commonly expressed by epithelial tumors of the skin, as well as epithelioid sarcoma and synovial sarcoma. 19*25~102~*03 Spurious instances of EMA reactivity in angiosarcoma have been reported; the lack of membrane-based staining-a pattern that is typical of truly epithelial neoplasms (Figure 6) -is a helpful clue in such cases.lo4 As noted above, muscle-specific isoforms of actin are useful determinants in the differential diagnosis of vascular tumors. The dermatopathologist must also be aware of the distribution of other actin subtypes. The latter are expressed by a variety of cells, including endothelial proliferations. Hence, one must be certain of the immuno-
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Figure 5. Vimentin in Epithelioid Angiosarcoma. Vimentin reactivity is present in the cytoplasm of virtually all neoplastic cells.
logic characteristics of anti-actins that are employed in diagnostic settings. Finally, the judicious use of antibodies to substances that are commonly expressed by melanocytic lesions will obviate concerns over the misdiagnosis of malignant melanoma as epithelioid vascular lesions. Of these, the substance recognized by HMB-45 is most characteristic of melanocytic neoplasms. The absence of reactivity for this antibody in most instances of spindled-desmoplastic melanoma should be remembered, however, when diagnostic considerations include the latter tumor, spindled angiosarcoma, spindle-cell hemangioendothelioma, and Kaposi’s sarcoma. lo5,106Antibodies to SlOO protein are helpful in distinguishing melanoma from these other lesions, inasmuch as they do not label vascular tumorsz5J07
Summary Immunohistochemistry is of proven value in the evaluation of cutaneous vascular neoplasms. Nonetheless, the lack of sensitivity and specificity demonstrated by some endothelial markers requires that a panel of antibodies to other lineage-related determinants be applied in this context. Compared with electron microscopy,12J3~10*-114 enzyme-linked antibody techniques can be applied in a cost-effective manner, with a satisfactory diagnostic result in most cases. The efficacy of these methods will likely improve as more sensitive and specific markers of endothelial differentiation are characterized. For the time being, the exclusion of non-endothelial proliferations re-
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2 991;9:243 - 253
Figure 6. Epithelial Membrane Antigen (EMA) in Adenoid (Acantholytic) Squamous Cell Carcinoma. Although this lesion may resemble epithelioid angiosarcoma, the accentuation of plasmalemma by antibodies to EMA facilitates its recognition as a “true“ epithelial neoplasm.
mains an important aspect of the differential diagnosis of vascular tumors.
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48. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:41115.
63. Carroll TJ, Schelper RL, Goeken JA, Kemp JD. Neoplastic angioendotheliomatosis: immunopathologic and morphologic evidence for intravascular malignant lymphoma. Am J Clin Path01 1986;85:169-75.
49. Little D, Said JW, Siegel RJ, et al. Endothelial cell markers in vascular neoplasms: An immunohistochemical study comparing factor VIII-related antigen, blood group specific antigens, 6-keto-PGFl alpha, and Ulex europaeus I lectin. J Path01 1986;149:89-95. 50. Ryan US, Ryan JW, Whitaker C, Chiu A. Localization of angiotensin converting enzyme (KININASE II). II. Immunocytochemistry and immunofluorescence. Tissue Cell 1976;8:125-45. 51. Schlingemann RO, Dingjan GM, Emeis JJ, et al. Monoclonal antibody PAL-E specific for endothelium. Lab Invest 1985;52:71-76. 52. Alles JU, Bosslet K. Immunohistochemical and immunochemical characterization of a new endothelial cell-specific antigen. J Histochem Cytochem 1986;34:209 - 14. 53. Bosslet K, Alles JU, Kayser K, et al. A small cell lung carcinoma cell line associated antigen (GP200) is a marker for human endothelial cells. Int Symposium on Peptide Hormones in Lung Cancer, Marburg, June 18-20, 1984. 54. Duijvestijn AM, Horst E, Pals ST, et al. High endothelial differentiation in human lymphoid and inflammatory tissues defined by monoclonal antibody HECA-452. Am J Path01 1988;130:147-55. 55. Fossum S, Smith ME, Ford WL. The migration of lymphocytes across specialized endothelium: VII. The migration of T and B lymphocytes from the blood of the athymic nude rat. Stand J Immunol 1983;17:539-50.
64. Fulling K, Gersell D. Neoplastic angioendotheliomatosis: Histologic, immunohistochemical and ultrastructural findings in two cases. Cancer 1983;51:1107-18. 65. Sheibani K, Battifora H, Winberg CD, et al. Further evidence that “malignant angioendotheliomatosis” is an angiotropic large-cell lymphoma. N Engl J Med 1986;314:943-48. 66. Wick MR, Mills SE, Scheithauer BW, et al. Reassessment of “malignant angioendotheliomatosis”: Evidence for its reclassification as “intravascular lymphomatosis”. Am J Surg Path01 1986;10:112-23. 67. Kurtin PJ, Pinkus GS: Leukocyte common antigen-a diagnostic discriminant between hematopoietic and nonhematopoietic neoplasms inparaffin sections using monoclonal antibodies. Hum Path01 1985;16:353-65. 68. Wick MR, Hagen KA, Frizzera G. Three immunostaining techniques for the localization of leukocyte common antigen in formalin-fixed, paraffin-embedded dermatologic biopsy specimens. Am J Dermatopathol 1987;9:250-55. 69. Berry CL, Amerigo J. Blood group antigens in vascular tumors. Evaluation of the immunoperoxidase technique. Virchows Arch (Path01 Anat) 1980;388:167-74. 70. Oriol R, Cooper JE, Davies DR, Keeling PWN. ABH antigens in vascular endothelium and some epithelial tissues of baboons. Lab Invest 1984;50:514-18.
56. Kaplan KL, Weber D, Cook P, et al. Monoclonal antibodies to E92, an endothelial cell surface antigen. Arteriosclerosis 1983;3:403-12.
71. Suzuki Y, Hashimoto K, Crissman J, et al. The value of blood group-specific lectin and endothelial associated antibodies in the diagnosis of vascular proliferations. J Cutan Path01 1986;13:40819.
57. Rutgers JL, Wieczorek R, Bonetti F, et al. The expression of endothelial cell surface antigens by AIDS-associated Kaposi’s sarcoma. Evidence for a vascular endothelial cell origin. Am J Path01 1986;122:493-99.
72. Weinstein RS, Coon J, Alrony J, et al. Tissue-associated blood group antigens in human tumors. In DeLellis RA (ed.) Diagnostic Immunohistochemistry. New York, Masson, 1984, pp 239-61.
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73. Wick MR, Kaye VN. The role of diagnostic immunohistochemistry in dermatology. Semin Dematol 1986;5:34658.
89. Stenback F, Wasenius VM, Kallioinen M. Basement membranes in experimentally-induced skin tumors. J Invest Dermatol 1986;87:185-89.
74. Feigl W, Denk H, Davidovits A, Holzner JH. Blood group isoantigens in human benign and malignant vascular tumors. Virchows Arch (Path01 Anat) 1976;370:323-32.
90. Listrom MB, Fenoglio-Preiser CM. Does laminin immunoreactivity really distinguish between lymphatics and blood vessels? Surg Path01 1988;1:71-76.
75. Holthofer H, Virtanen I, Kariniemi AL, et al. Ulex europaeus I lectin as a marker for vascular endothelium in human tissues. Lab Invest 1982;47:60-66.
91. Choy M-Y, Richman PI, Horton MA, MacDonald TT. Expression of the VLA family of integrins in human intestine. J Path01 1990;160:35-40.
76. Miettinen M, Holthofer H, Lehto VP, et al. Ulex europaeus I lectin as a marker for tumors derived from endothelial cells. Am J Clin Path01 1983;79:32-36.
92. Holzmann B, McIntyre BW, Weissman IL. Identification of a murine Peyer’s patch-specific lymphocyte homing receptor as an integrin molecule with an alpha chain homologous to human VLA-4-alpha. Cell 1989;56:37-46.
77. Ordonez NG, Batsakis JG. Comparison of Ulex europaeus I lectin and factor VIII-related antigen in vascular lesions. Arch Path01 Lab Med 1984;108:129-32. 78. Stadler R, Schaumburg-Lever G, Orfanos CE. Evaluation of benign and malignant endothelial proliferations using Ulex europaeus I as an endothelial marker (abstr). Arch Dermatol 1984;120:1616. 79. Pober JS, Gimbrone MAJ. Expression of Ia-like antigens by human vascular endothelial cells in vitro: Demonstration by monoclonal antibody binding and immunoprecipitation. Proc Nat1 Acad Sci USA 1982;79:6641-45. 80. Swanson PE, Wick MR. HLA-DR (Ia-like) reactivity in tumors of bone and soft tissue. An immunohistochemical comparison of monoclonal antibodies LN3 and LK8D3 in routinely-processed specimens. Mod Path01 1990;3: 113 19. 81. Beckstead JW, Wood GS, Fletcher V. Evidence for the origin of Kaposi’s sarcoma from lymphatic endothelium. Am J Path01 1985;119:294-300. 82. Harrison AC, Kahn LB. Myogenic cells in Kaposi’s sarcoma: An ultrastructural study. J Path01 1978;124:15760. 83. McNutt NS, Fletcher V, Conant MA. Early lesions of Kaposi’s sarcoma in homosexual men. An ultrastructural comparison with other vascular proliferations in skin. Am J Path01 1983;111:62-77. 84. Auerbach HE, Brooks JJ. Kaposi’s sarcoma: Neoplasia or hyperplasia? Surg Path01 1989;2:19-25. 85. Barsky SH, Baker A, Siegal GP, et al. Use of anti-basement membrane antibodies to distinguish blood vessel capillaries from lymphatic capillaries. Am J Surg Path01 1983;7:667-77. 86. Ekblom I’, Miettinen M, Rapola J, Foidart J-M. Demonstration of laminin, a basement membrane glycoprotein, in routinely processed formalin-fixed human tissues. Histochemistry 1982;75:301-07. 87. Lee AK. Basement membrane and endothelial antigens: Their role in the evaluation of tumor invasion and metastasis. In DeLellis RA (ed.) Advances in Immunohistochemistry. Raven Press, New York, 1988, pp 363-93. 88. Miettinen M, Foidart JM, Ekblom P. Immunohistochemical demonstration of laminin, the major glycoprotein of basement membranes, as an aid in the diagnosis of soft tissue tumors. Am J Clin Path01 1983;79:30611.
93. Altmannsberger M, Dirk T, Osbom M, Weber K. Immunohistochemistry of cytoskeletal filaments in the diagnosis of soft tissue tumors. Semin Diagn Path01 1986;3:306-16. 94. Miettinen M, Lehto VP, Badley RA, Virtanen I. Expression of intermediate filaments in soft tissue sarcomas. Int J Cancer 1982;30:541-46. 95. Osbom M, Altmannsberger M, Debus E, Weber K. Differentiation of the major human tumor groups using conventional and monoclonal antibodies specific for individual intermediate filament proteins. Ann NY Acad Sci 1985;455:649-68. 96. Swanson PE, Stanley MW, Scheithauer BW, Wick MR. Primary cutaneous leiomyosarcoma. A histological and immunohistochemical study of 9 cases, with ultrastructural correlation. J Cutan Path01 1988;15:129-41. 97. Tsukuda T, McNutt MA, Ross R, Gown AM. HHF35, a muscle actin-specific monoclonal antibody. II. Reactivity in normal, reactive, and neoplastic human tissue. Am J Path01 1987;127:389-402. 98. Eusebi V, Carcangiu ML, Dina R, Rosai J. Keratin-positive epithelioid angiosarcoma of thyroid (abstr). Lab Invest 1990;62:32A. 99. Gray MH, Rosenberg AE, Dickersin GR, Bhan AK. Cytokeratin expression in epithelioid vascular neoplasms. Hum Path01 1990;21:212-17. 100. Palman C, Brooks JJ, LiVolsi VA. Aberrant expression of cytokeratin in vascular tissues and tumors (abstr). Lab Invest 1990;62:77A. 101. van Haelst UJGM, Pruszczynski M, ten Cate LN. Ultrastructural and immunohistochemical study of epithelioid hemangioendothelioma of bone: Coexpression of epithelial and endothelial markers. Ultrastruct Path01 1990; 14:141-49. 102. Pinkus GS, Kurtin PJ. Epithelial membrane antigen-a diagnostic discriminant in surgical pathology. Hum Path01 1985;16:929-40. 103. Sloane JP, Ormerod MG. Distribution of epithelial membrane antigen in normal and neoplastic tissues and its value in diagnostic pathology. Cancer 1981;47:1786-95. 104. Swanson PE, Manivel JC, Scheithauer BW, Wick MR. Epithelial membrane antigen reactivity in mesenchymal neoplasms: An immunohistochemical study of 306 soft tissue sarcomas. Surg Path01 1989;2:313-22.
Clinics in Dermatology 1991;9:243 - 253 105. Gown AM, Vogel AM, Hoak D, et al. Monoclonal antibodies specific for melanocytic tumors distinguish subpopulations of melanocytes. Am J Path01 1986;123:195-203.
106. Wick MR, Swanson PE, Rocamora A. Recognition of malignant melanoma by monoclonal antibody HMB-45. An immunohistochemical study of 200 paraffin-embedded cutaneous tumors. J Cutan Path01 1988;15:201-07.
107. Weiss SW, Langloss JM, Enzinger FM. Value of S-100 protein in the diagnosis of soft tissue tumors with particular reference to benign and malignant schwann cell tumors. Lab Invest 1983;49:299-311.
108. Capo V, Ozzello L, Fenoglio CM. Angiosarcomas arising in edematous extremities: Immunostaining for factor VIIIrelated antigen and ultrastructural features. Hum Path01 1985;16:144-50.
109. Carstens PHB. The Weibel-Palade
body in the diagnosis of endothelial tumors. Ultrastruct Path01 1981;2:315-25.
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110. Henderson DW, Papadimitriou JM, Coleman M (eds). Ultrastructural Appearances of tumours, Second Edition. New York, Churchill Livingstone, 1986, pp 246-54. 111. Llombart-Bosch A, Peydro-Olaya A, Pellin A. Ultrastructure of vascular neoplasms. Path01 Res Pratt 1982;174:141. 112. Mackay B, Osborne BM. The contribution of electron microscopy to the diagnosis of tumors. Pathobiol Annu 1978;8:359-405. 113. Meister P, Hoede N, Rumpelt HJ. Epithelioid hemangioendothelioma of the scalp: A case report with immunohistochemical and ultrastructural study. Path01 Res Pratt 1985;180:220-26. 114. Waldo ED, Vuletin JC, Kaye GI. The ultrastructure of vascular tumors: Additional observations and a review of the literature. Path01 Annu 1977;12:179-308.
E. SWANSON, R. WICK,
MD
MD
he histologic features of vascular neoplasms arising in the superficial soft tissue and skin are considerably more complex than one might predict from the appearance of the normal endothelial cell. This attribute of endothelial proliferations may cause substantial diagnostic confusion in some instances. The most troublesome to the pathologist are those neoplasms in which a spindled or an epithelioid population predominates. Because spindle cell tumors of the skin are addressed elsewhere in this monograph, much of the ensuing discussion will focus on overtly vasogenic neoplasms and their epithelioid histologic variants. The purpose of this presentation is not to deal with these entities in a comprehensive manner, because their morphologic features have been discussed at length elsewhere.1-20 Instead, we will review the application of immunohistochemical techniques to the routine evaluation of vascular proliferations. The fundamentals of immunohistochemistry are no longer foreign concepts to most practitioners of dermatopathology, nor is there much disagreement as to the relative efficacy of the various methodologies that are available. These include the peroxidase anti-peroxidase (PAP)** and the avidin-biotin-peroxidase complex (ABC) techniques** and variations or modifications thereof.23 In most settings, we prefer the latter procedure, although most of the antibodies applicable to the evaluation of endothelial differentiation are not dependant on methodologic choices. The greatest advantage of the ABC technique is that its relative efficiency (compared to PAP) has obviated the need for certain modifications designed to enhance the sensitivity of the assay. Various aldehyde
T
From the Department of Pathology, Washington University School of Medicine, St. Louis, Missouri. Address correspondence to: Paul E. Swanson, M.D., Division of Anatomic Pathology, Barnes Hospital, 2 Barnes Hospital Plaza, St. Louis MO
63110.
0 2991 by Elsevier Science Publishing
Co., Inc.
l
0738-081x/91/$3.50
and alcohol-based fixatives have been employed to improve the preservation of endothelial antigens,24*25 but in many instances, routinely processed tissues yield readily interpretable immunohistochemical results. Similarly, the use of protease digestion, to “unmask’ fixation-altered cellular antigens, was historically considered a requirement in the immunohistochemical detection of some endothelial determinants.25-27 With current commercial ABC products and antibody reagents, however, this manipulation has become unnecessary in most cases. Various cell products have been described (in addition to vWF), which, when detected immunohistochemically, point towards endothelial differentiation. The conjoint use of these markers often simplifies the diagnostic problems posed by vascular neoplasms, a concept that is amplified in the ensuing discussion and summarized in Tables 1 and 2.
Factor VIII-Related Antigen and Thrombomodulin Despite the unique functions performed by endothelial cells, relatively few biological markers consistently label neoplastic endothelium. The emphasis on the term neoplastic is important, because several cell products elaborated by normal or reactive endothelium are underexpressed, or, for practical purposes, are absent in a large proportion of benign and malignant vascular tumors. Perhaps the best known of these substances is von Willebrands factor (vWF : Factor VIII-related antigen). This clotting co-factor is actively synthesized by virtually all non-lymphatic endothelial cells,2**29 and is probably stored in and transported by the Weibel-Palade bodies that ultrastructurally characterize such cells.30 Commercially available monoclonal and polyclonal antibodies with proven specificity for vWF have been successfully applied to the immunohistochemical analysis of routinely processed tissues. Nonetheless, the diagnostic value of
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2991;9:243-253 Table 1. Immunohistochemisty
in the Differential Diagnosis of Hemanxioma
vWF
Tumor Hemangioma Lymphangioma Glomus Tumor Hemangiopericytoma Angiomatoid Fibrous Histiocytoma Cellular Angiolipoma Spitz Nevus Spiradenoma
UEA
TM
+/0 0 0 0
+ + 0 0 0
+ 0 0
+ 0 0
mACT
CK
EMA
HMB45
SIOO
+ + +/+ 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
0 0 0 0 0
+ 0 0
0 0 0
0 0 +
0 0 +
0 + 0
+ + +
C4L
ACT
+ 0 0 0 0
+ 0 + +/0
0 nt nt
+/0 0
Abbreviations: vWF= von Willebrand’s factor (Fnctor VIII-related antigen); UEA = Ulex europaeus agglutinin I; TM = thrombomodulin; C4L = collagen type ZV/laminin; ACT = actin; mACT = muscle-specific actin; CK = cytokeratin; EMA = epifhelial membrane antigen; nt = not tested.
these antibodies in the recognition of vascular neoplasia is limited.31 Indeed, even when protease digestion and other enhancement techniques are applied, only half of benign vascular lesions are reproducibly labeled for vWF, and fewer than 25% of malignant endothelial lesions (Figure 1) are similarly immunoreactive.26~27~31-37 Nonetheless, antibodies to vWF are a specific marker of endothelial differentiation, and accordingly remain an important part of the immunohistochemical diagnosis of vascular tumors. Expectedly, neoplasms that contain predominantly non-endothelial vasofonnative elements, including glomus tumor and hemangiopericytoma, are typically negative for vWF.25,38,39 Somewhat better results have been obtained using antibodies to the endothelial-selective product thrombomodulin (TM).40,41 This substance, which is essentially an antagonist of vWF, acts by forming a stable stoichiometric complex with thrombin, which, in turn, manifests enhanced activation of the anticoagulant protein C. In contrast to vWF, TM is more strongly expressed in malignant endothelial proliferations. In one published series, polyclonal antibodies to TM labeled each of 14 angiosarco-
Table 2. lmmunohistochemisty
vWF
Tumor Epithelioid
Angiosarcoma
in the Differential
and
mas, as well as examples of epithelioid hemangioendothelioma, and various benign hemangiomas.41 Notably, neither the glomus cells nor the endothelial cells of glomus tumor were convincingly stained. The reciprocal relationship between the expression of vWF and TM in vascular tumors has not been well-explained, although the impact it may have on the absence of effective hemostasis in malignant endothelial lesions has been the subject of some conjecture. 41 The greatest weakness of antibodies to this marker is that TM-reactive substances are also elaborated by certain non-endothelial neoplasms, including squamous cell carcinoma41 and trophoblastic neoplasms. 42 The former is an important concern, because of the close histologic similarities that may exist between epithelioid angiosarcoma and the so-called acantholytic variant of squamous cell carcinoma.43 Further studies of endothelial and non-vascular neoplasms may help to define the role of this marker. Unfortunately, antibodies to TM are not yet commercially available. Antibodies have been raised to a family of endothelial products called endothelins. I4 However, these vasoactive oligopeptides are not selectively expressed by endothe-
Diagnosis of Epithelioid
TM
UEA
Endothelial
DES/
Neoplasms
CV
mACT
EMA
HMB
SIOO
LCA
DR
C4L
+/-
+
+
0
0
0
0
0
0
+
Hemangioendothelioma Epithelioid Sarcoma Epithelioid Leiomyosarcoma Malignant Lymphoma (including “malignant angioendotheliomatosis”)
0 0 0
nt 0 0
+/0 0
0 + 0
+ 0t 0
0 0 0
0’ 0t 0
0 0 +
+/+/+
0 + 0
Metastatic Carcinoma Adenoid Squamous Cell Carcinoma Malignant Melanoma
0 0 0
nt +/0
+/0 0
0 0 0
+ + 0
0 0 +
+/0 +
0 0 0
+/0 +
+/0 0
Abbreviations: uWF = van Willebrand’sfuctor; TM = thrombomodulin; UEA = UIex europaeus agglufinin I; DES = desmin; mACT = muscle-specific actin; CK = cytokerafin; EMA = epithelial membrane antigen; LCA = leukocyte common antigen; DR = P&l-DR antigen; C4L = collagen type IV/Zaminin; nt = not tested. l Occasional cases nre SIOO-positive. f Occasional cutaneous leiomyosnrcomas may be reactive for CK, EMA, and SIOO.
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Figure 1. von Willebrand Factor (vWF: Factor VIII-related Antigen) in Angiosarcoma. lmmunoreactivity for vWF is localized to endothelial cytoplasm in fewer than 25% of angiosarcomas.
lium, and are thus of little value as immunohistochemical markers.45-49 Similar comments apply to the detection of other endothelial-associated products, such as 5’-nucleotidase, 6-keto-prostaglandin-F1-alpha,49 antithrombin 111, thrombospondm-and angiotensin converting enzyme.50
Monoclonal Antibodies to Other EndotheliumSelective Determinants Considerable effort has been expended in the production of monoclonal antibodies to intact and cultured endothelial cells or cell lysates. Of these, the antibodies PAL-E51,* EN-4,24 and BW20052,53 have received the most attention. The greatest drawback to the routine application of certain of these antibodies is their requirement for frozen or *Actually, PAL-E was one of several antibody clones produced by a mouse thaf had been immunized with cells recovered from a lymph nodal metasfasis of malignant melanoma. 51 We can only assume that endothelial cells contaminating the preparation were also injected.
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specially prepared tissue substrates. Among the latter reagents, only BW200 exhibits reliable reactivity in formalin-fixed, paraffin-embedded tissues. Nonetheless, given the availability of appropriate tissues, these antibodies are of potential value for two reasons. One is their putative specificity for endothelial cells. The second is the selective non-reactivity with endothelial lymphatics exhibited by PAL-E.51 The application of a panel of these markers, together with antibodies to vWF and TM, may conceivably distinguish between lesions of vascular or lymphatic endothelial origin. The practical use of this information is limited, however, and is likely to be of little consequence to the clinical diagnosis and treatment of angiosarcoma. The same concerns may be voiced regarding the applicability of the antibody HECA-452, a monoclonal product with putative specificity for cells differentiating as postcapillary “high endothelium.” 54 These reagents are of greater interest to those exploring the differentiation and function of normal endothelium, and its interaction with lymphoid and extravascular tissues.55 Several other antibodies with selectivity for endothelium have been described, including E92,56,57 HC-1,58 0KM-1,59 and 0KM-5.59 In each instance, however, significant reactivity also has been noted with hematolymphoid or non-endothelial mesenchymal cell populations, setting the stage for the inadvertant misinterpretation of an intra- or perivascular lymphoid proliferation as endothelial in nature. Tumors with affinity for lymphoid elements, such as the endovascular papillary angioendothelioma,3*60*61or others composed of malignant lymphoid cells, such as intravascular lymphomatosis (“malignant angioendotheliomatosis”)62-66 are perhaps the more important examples of this potential interpretive pitfall. In each case, antibodies directed at lymphoid-specific determinants, such as leucocyte common antigen, or B- and T-lineage selective antigens,67,68 will correctly identify the lymphoid elements in such lesions.
Blood Group-Related Antigens The expression of type-specific ABO blood group antigens is a normal function of non-lymphatic endothelium. In neoplastic alterations, host type-specificity may be lost, but ABO substances persist in most instances.69-73 It follows from this observation that antibodies to A, B, and H antigen may be of value in the detection of endothelial differentiation. Specifically, antibodies to H-substance have been shown to label the majority of benign and malignant endothelial proliferations.69,74 Unfortunately, as with other markers discussed above, the presentation of ABO antigens is not a unique function of endothelium among non-hematopoietic tissues. Normal and neoplas-
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Figure 2. Ulex europaeus 1 agglutinin (LIE& in Epithelioid angiosarcoma. More commonly identified than vWF, UF.A crisply labels endothelial cell surfaces.
tic epidermal and cutaneous adnexal epithelium, as well as some non-vascular sarcomas, may also produce immunodetectable H antigen.25~69~70~72~73 These comments are of considerable importance to our understanding of the utility of the plant lectin Ulex europaeus I agglutinin (UEA) in the histochemical evaluation of vascular tumors.75 Although the ability of UEA to recognize and bind to terminal fucosyl residues allows it to label a variety of glycoproteinaceous substances, the most common of the latter in human tissues is the H antigen. As a result, this lectin, in a manner similar to that of antibodies to H substance, is capable of recognizing a variety of non-vascular neoplasms, including certain adenocarcinomas and epithelioid sarcoma, each of which may be occasionally considered in the differential diagnosis of epithelioid vascular lesions.69,70,72*73Nonetheless, the striking sensitivity of this marker for endothelial malignancies (greater than 90%)76-78 proves its value as a marker for vascular differentiation (Figure 2) when an immunohistochemical analysis otherwise excludes other diagnostic alternatives. Together with antibodies to vWF, this lectin is useful in the immunohistochemical evaluation of cutaneous and soft tissue neoplasms. Antibodies to human histocompatibility antigens, including HLA-A,B,C, and beta-2-microglobulin (Class I) and HLA-DR (Class II), are of limited value in the diagnostic evaluation of endothelial tumors (and human neoplasia in general). Class I antigens are widely expressed by endothelium, mesenchyme and virtually all epithelial tissues, and induction of Class II antigens also may be
Clinics in Dermatology 1992:9:243-253 observed in a variety of reactive and neoplastic disorders of these tissues.7*,79,80 Nonetheless, the demonstration of HLA-DR in an epithelioid malignancy of skin may be of some practical use, as examples of angiosarcoma studied to date have not been immunoreactive for these determinants, whereas epithelioid sarcoma and malignant melanoma may be HLA-DR positive (Figure 3).*O Interestingly, Kaposi’s sarcoma may occasionally contain HLA-DR reactive cells, as well.sO The latter observation, taken together with the absence of reactivity for ABH antigens, and, in our experience, only sporadic or unreliable reactivity for vWF,*~ has provided suggestive immunohistochemical evidence that a fundamental difference exists between Kaposi’s sarcoma and other endothelial neoplasms as a group. There is, of course, considerable disagreement on this and at least one recent analysis of KS topic, 7*32,34-37*81-*3 purports to show both vWF and UEA staining in a majority of cases. ~4Nonetheless, the general lack of consistency in the staining characteristics of KS in other studies emphasizes the capricious nature of this neoplasm, and underscores our current inability to understand it fully using routinely available techniques. A similar problem has been encountered in the evaluation of the recently char“spindle-cell hemangioendothelioma” acterized (SCH).15J7 Although this tumor is clearly vasogenic and shows areas resembling cavernous hemangioma and epithelioid hemangioendothelioma, the ultrastructural and immunophenotypic characteristics of the spindled cells in SCH are not clearly endothelial in nature. The lack of vWF staining in the latter cells in the few examples studied to date,15 suggests that innately endothelial proliferations may have the capacity to evolve into cell populations with indeterminate or non-endothelial mesenchyma1 differentiation. Further study of Kaposi’s sarcoma and SCH may help to substantiate this hypothesis.
Matrical Proteins and Matrical Binding Proteins A consistent feature of normal vascular endothelium is its ability to elaborate an intact basement membrane; this function is at least partially preserved in neoplastic proliferations as well. Antibodies to the matrical components collagen type IV and laminin have been applied to the diagnosis of vascular neoplasms.71,85-*9 While there have been insufficient data presented with which to evaluate their overall sensitivity for endothelial lesions, it should be evident that they are non-specific. Even so, the expression of these substances by vascular neoplasms has been tested in two specific settings. First, it has been suggested that neither type IV collagen nor laminin is typically produced by lymphatic endothelium or its neoplasms, and like PAL-E, they may aid in the separation of
Clinics in Dermatology 2991;9:243 - 253
Figure 3. Class I1 Histocompatibility Antigens in Epithelioid Malignancies. Epithelioid angiosarcoma does not express HLADR (left), whereas histologically similar examples of malignant lymphatic from vascular lesions in skin and other sites (Figure 4).a’ However, the recent demonstration of laminin reactivity in both capillaries and lymphatics in gastric tissue calls this general assumption into question.90 Second, several authors have suggested that matrical proteins are present to a quantifiably lesser degree in angiosarcoma and Kaposi’s sarcoma, compared to benign vascular neoplasms. ‘~JX Whereas this postulate may have biochemical and morphologic support, there is no compelling evidence that immunohistochemistry can reliably detect useful differences in this regard. Attempts to establish the malignancy of a neoplasm on the basis of immunohistochemical staining are a tenuous and unwise pursuit at best; the proposition that matrical proteins might serve such a purpose only promulgates a dangerous fallacy. Another approach to the identification of vascular tumors is the detection of matrical ligand-binding substances at the endothelial cell surface. Perhaps the best understood elements among cell adhesion proteins are the integrins, a complex but interrelated group of heterodimeric molecules with relative specificity for different
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melanoma may be reactive (right), using the monoclonal antibody LN3.
matrical peptides. 91 No one integrin is specific for endothelium, although the presence of the integr-in VIA-4 or similar proteins in part characterizes certain lymphoidassociated endothelial cell populations.92 For reasons described above, these proteins are of considerable interest to students of endothelial cell function, but they have little application to diagnostic immunohistochemistry.
Non-Endothelial Markers of Importance in the Diagnosis of Vascular Lesions As the foregoing discussion implies, the immunohistochemical recognition of endothelial tumors usually relies on information other than reactivity for endothelial-related antigens. Bearing in mind that the lack of such reactivity may in part be the result of fixation-induced loss or alteration of these determinants, any immunohistochemica1 analysis of a given neoplasm requires an assessment of the presence of a substance known to be expressed. Our recommendation along these lines is that antibodies to vimentin be applied, in order to evaluate staining of mesenchymal elements. One of the latter, of
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Clinics in Dermatology 1991;9:243-253
Figure 4. Matrical Proteins in Benign Vascular Tumors. Antibodies to laminin stain the basement membrane around
course, is endothelium, which contains vimentin as its principal structural protein. Not surprisingly, virtually all vascular neoplasms, unless handled improperly during processing, are vimentin-reactive (Figure 5).25*93-95 Other intermediate filaments of interest in the evaluation of endothelial tumors are desmin and cytokeratin, not because they are normally expressed by endothelium, but because their detection generally excludes the diagnosis of endothelial differentiation. The former of these moieties is characteristic of normal and neoplastic myogenie elements,93-95 and together with muscle-specific actin isoforms, constitutes the basis for the detection of muscle differentiation in cutaneous neoplasms.25,96*97 Cytokeratins serve a similar function, as their presence typically equates with epithelial differentiation. Reactivity for these peptides in primary and metastatic carcinomas of the skin, and epithelioid sarcoma,24,25*93-95 remains the most convincing indication that the alternative diagnosis of an epithelioid vascular neoplasm is incorrect. Recent reports, however, have questioned this conclusion. In four separate studies, immunoreactivity for low-molecular weight cytokeratins has been described in angiosarcoma and epithelioid hemangioendothelioma.98-10* At
neoplastic endothelial cells in cavernous hemangioma usually do not stain lymphangiomas (right).
(left), but
this point, biochemical demonstration of cytokeratins in these tissues is lacking. Moreover, evaluation of the methodologic aspects of such studies suggests that the cytokeratin “positivity” they report is unreliable, in that it was obtained only at high antibody concentration. In light of these claims, a more extended panel of antibodies to epithelial determinants may provide some solace to the observer who is unwilling to rely on cytokeratins in this setting. Epithelial membrane antigen (EMA) and carcinoembryonic antigen, among others, represent determinants that are commonly expressed by epithelial tumors of the skin, as well as epithelioid sarcoma and synovial sarcoma. 19*25~102~*03 Spurious instances of EMA reactivity in angiosarcoma have been reported; the lack of membrane-based staining-a pattern that is typical of truly epithelial neoplasms (Figure 6) -is a helpful clue in such cases.lo4 As noted above, muscle-specific isoforms of actin are useful determinants in the differential diagnosis of vascular tumors. The dermatopathologist must also be aware of the distribution of other actin subtypes. The latter are expressed by a variety of cells, including endothelial proliferations. Hence, one must be certain of the immuno-
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Figure 5. Vimentin in Epithelioid Angiosarcoma. Vimentin reactivity is present in the cytoplasm of virtually all neoplastic cells.
logic characteristics of anti-actins that are employed in diagnostic settings. Finally, the judicious use of antibodies to substances that are commonly expressed by melanocytic lesions will obviate concerns over the misdiagnosis of malignant melanoma as epithelioid vascular lesions. Of these, the substance recognized by HMB-45 is most characteristic of melanocytic neoplasms. The absence of reactivity for this antibody in most instances of spindled-desmoplastic melanoma should be remembered, however, when diagnostic considerations include the latter tumor, spindled angiosarcoma, spindle-cell hemangioendothelioma, and Kaposi’s sarcoma. lo5,106Antibodies to SlOO protein are helpful in distinguishing melanoma from these other lesions, inasmuch as they do not label vascular tumorsz5J07
Summary Immunohistochemistry is of proven value in the evaluation of cutaneous vascular neoplasms. Nonetheless, the lack of sensitivity and specificity demonstrated by some endothelial markers requires that a panel of antibodies to other lineage-related determinants be applied in this context. Compared with electron microscopy,12J3~10*-114 enzyme-linked antibody techniques can be applied in a cost-effective manner, with a satisfactory diagnostic result in most cases. The efficacy of these methods will likely improve as more sensitive and specific markers of endothelial differentiation are characterized. For the time being, the exclusion of non-endothelial proliferations re-
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Figure 6. Epithelial Membrane Antigen (EMA) in Adenoid (Acantholytic) Squamous Cell Carcinoma. Although this lesion may resemble epithelioid angiosarcoma, the accentuation of plasmalemma by antibodies to EMA facilitates its recognition as a “true“ epithelial neoplasm.
mains an important aspect of the differential diagnosis of vascular tumors.
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