Monoclonal Antibody D2-40, a New Marker of Lymphatic Endothelium, Reacts with Kaposi’s Sarcoma and a Subset of Angiosarcomas Harriette J. Kahn, M.D., Denis Bailey, M.D., Alexander Marks, M.D. Department of Pathology, Women’s College Campus, Sunnybrook and Women’s College Health Sciences Centre (HJK, AM); Department of Pathology (DB), University Health Network, Banting; and Best Department of Medical Research (AM), University of Toronto, Toronto, Canada omas, in which staining was absent. We conclude that There is controversy over the histogenesis of Kaposi’s D2-40 is a new selective marker of lymphatic endo- sarcoma (KS) from lymphatic or blood vessel endo- thelium in normal tissues and vascular lesions and is thelium. D2-40 is a novel monoclonal antibody to an valuable for studying benign and malignant vascular Mr 40,000 O-linked sialoglycoprotein that reacts with disorders in routinely processed tissue specimens. a fixation-resistant epitope on lymphatic endothe- lium. We sought to establish the selectivity of D2-40 KEY WORDS: Kaposi’s sarcoma, Lymphatic endo- for lymphatic endothelium in normal tissues and thelium, Monoclonal antibody, Vascular lesions. compare its reactivity with the expression of the Mod Pathol 2002;15(4):434–440 widely used vascular endothelial marker CD31 in a series of 62 formalin-fixed and paraffin-embedded Kaposi’s sarcoma (KS) is a vascular proliferative vascular lesions including KS. In normal tissues, disorder with four epidemiological forms: classic, D2-40 stained the endothelium of lymphatic channels African endemic, iatrogenic transplant related, and but not of blood vessels, including arteries and capil- epidemic acute immune deficiency syndrome laries defined by reactivity with the blood vessel endo- (AIDS) related (1). KS is usually limited to the skin thelial marker PAL-E. In our series of vascular lesions, but may involve mucus membranes, visceral or- D2-40 stained lymphangiomas (10/10), benign tumors gans, and lymph nodes in the more aggressive of undisputed lymphatic origin, but not benign neo- forms (2). For cutaneous KS, a histopathological plasms or tumorlike lesions of blood vessel origin, progression from patch to plaque and nodular including hemangiomas (0/10), glomus tumors (0/3), stages correlates with a clinical progression from angiolipomas (0/2), pyogenic granulomas (0/2), vascu- macules to palpable tumors (2). The patch stage is lar malformations (0/2), hemangiopericytoma (0/1), characterized by networks of thin-walled irregular or hemangioendothelioma (0/1). D2-40 stained all endothelial-lined spaces; the plaque stage, by cases of cutaneous KS (24/24) at all stages of progres- sparse spindle cells intermingled among irregular sion, including patch, plaque, and nodular stages, endothelial-lined spaces; and the nodular stage, by supporting the concept that this disease originates sheets or fascicles of spindle cells (2). from a cell type capable of undergoing lymphatic dif- Although the exact pathogenesis of KS is not de- ferentiation. D2-40 also stained three of seven angio- fined, it has been suggested that a chronic cytokine- sarcomas, indicating that a subset of these tumors can mediated proliferative state of an endothelial cell, undergo at least partial differentiation along the lym- phatic endothelial lineage and could be classified as possibly triggered by activation of a latent KS- lymphangiosarcomas. In comparison, CD31 was ex- associated herpes virus (KSHV) infection, promotes pressed in all benign and malignant vascular lesions, transformation to a neoplastic cell (1, 3, 4). The origin except for glomus tumors (0/3) and 5/10 lymphangi- of KS from blood vessel or lymphatic endothelium is controversial. Although some early studies failed to detect the presence of blood vessel endothelial mark- ers in KS (5), other studies reported the presence of Copyright © 2002 by The United States and Canadian Academy of Pathology, Inc. traditional blood vessel endothelial markers including VOL. 15, NO. 4, P. 434, 2002 Printed in the U.S.A. CD31, CD34, and von Willebrand factor (vWF, Factor Date of acceptance: December 6, 2001. Address reprint requests to: Harriette J. Kahn, M.D., Department of Pa- VIII–related antigen) (6–9). The latter findings are thology, Sunnybrook and Women’s College Health Sciences Centre, Sun- nybrook Campus, E4-33, 2075 Bayview Avenue, Toronto, Ontario, Can- compatible with a theory of origin of KS from blood ada, M4N 3M5; fax: (416) 480-4271; e-mail: [email protected]. vessel endothelium. However, this interpretation is 434 complicated by recent observations that the above (Hornby, Ontario, Canada). The mAb to CD31 blood vessel endothelial markers are also weakly ex- (Clone JC/70A, hybridoma supernatant) was pur- pressed on lymphatic endothelium (9–11). Further- chased from DAKO (Carpinteria, CA). more, ultrastructural features and the absence of the Sections of paraffin-embedded tissues (5 ␮m) were blood vessel endothelium–specific PAL-E antigen (12) dewaxed through graded concentrations of ethanol. from KS argue against an origin from blood vessel For immunostaining, heat-inducted epitope retrieval endothelial cells (13–15). These considerations have by microwave pretreatment (800 W for 10 min) was led some authors to suggest that KS originates from only performed for CD31. The sections were first in- lymphatic endothelium (5, 14). This notion has been cubated in methanol containing 3% H2O2 to inacti- reinforced recently by the demonstration of a uniform vate endogenous peroxidase. The sections were then expression of two lymphatic endothelial markers, vas- incubated with mAb D2-40 (0.1 ␮g/mL), or anti-CD31 cular endothelial growth factor receptor-3 (VEGFR-3; (1:40 dilution), followed sequentially by biotinylated 16) and podoplanin (17), in KS (18–20). However, goat anti-mouse immunoglobulin (IgG) antibody VEGFR-3 has also been shown to be expressed by (Zymed, San Francisco, CA) at a 1:200 dilution and a neoplastic blood vessel endothelia in both benign and horseradish peroxidase–avidin conjugate (DAKO) at a malignant vascular tumors and, in this situation, loses 1:500 dilution. For color development, the sections its selectivity for lymphatic endothelium (21). There- were incubated with 3,3'-diamino-benzidine. Adja- fore, we wished to reexamine these results with an cent cryostat sections (5 ␮m) of quick-frozen tissues independent marker of lymphatic endothelium. were air-dried and fixed in cold acetone for 10 min. We recently observed that D2-40, a monoclonal After inactivation of the endogenous peroxidase as antibody (mAb) to an Mr 40,000 O-linked sialogly- described above, the sections were immunostained coprotein (22), was a selective marker of lymphatic with mAb PAL-E (0.15 ␮g/mL) or D2-40 (0.1 ␮g/mL), endothelium. In this study, we examined the ex- also as described above. pression of this novel marker of lymphatic endo- thelium in normal tissues and compared its reac- tivity with the expression of CD31 in a series of 62 RESULTS vascular lesions, including 24 cases of cutaneous KS at different stages of progression. Consecutive cryostat sections of human tonsil were stained with mAb D2-40 or PAL-E (that is specific for endothelial cells of blood vessels; 12). MATERIALS AND METHODS These antibodies stained nonoverlapping popula- tions of thin-walled endothelial channels in adja- Formalin-fixed, paraffin-embedded blocks of cent sections. PAL-E stained blood vessels (Fig. 1A, normal tissue, 62 vascular lesions (24 cutaneous KS, arrowheads) that did not stain with D2-40 (Fig. 1B). 7 angiosarcomas, 10 lymphangiomas, 10 hemangi- Conversely, D2-40 stained lymphatic channels (Fig. omas, 3 glomus tumors, 2 angiolipomas, 2 pyogenic 1B, arrowheads) that did not stain with PAL-E (Fig. granulomas, 2 vascular malformations, 1 heman- 1A). In paraffin sections of normal breast, D2-40 giopericytoma, 1 hemangioendothelioma), 28 non- stained the endothelium of lymphatic channels vascular soft-tissue tumors (10 leiomyosarcomas, 6 (Fig. 1C, arrowheads), but not the endothelium of neurofibromas, 6 dermatofibromas, 4 dermatofi- an artery in a vascular bundle (Fig. 1C, arrow). brosarcoma protuberans, 2 malignant fibrous histi- D2-40 also stained the endothelium of a lym- ocytomas), as well as frozen samples of tonsil, were phatic channel containing lymphocytes (Fig. 1D, obtained from the Department of Pathology, Sun- arrowhead), but not the adjacent capillary (Fig. 1D, nybrook and Women’s College Health Sciences arrow). The results of immunostaining of a series of Center, University of Toronto, Ontario, Canada. vascular lesions with mAb D2-40 and for CD31 are The KS specimens included patch, plaque, and summarized in Table 1. D2-40 reacted strongly with nodular lesions from patients with classic and all cases of cutaneous KS (24/24) at the patch (Fig. AIDS- and transplant-related KS. None of the an- 2B–C), plaque, and nodular (Fig. 2E–F) stages of giosarcomas were associated with lymphedema or progression. D2-40 also reacted with the endothe- preexisting lymphangioma. lium lining the sinusoidal spaces in all lymphangi- MAb D2-40 (IgG1) was purified from mouse as- omas (10/10; Fig. 3A) but did not react with any citic fluid, as previously described (22), and stored hemangiomas (0/10), glomus tumors (0/3), angio- at a concentration of 0.7 mg/mL under sterile con- lipomas (0/2), pyogenic granulomas (0/2), vascular ditions at 4° C. The mAb to an undefined blood malformations (0/2), hemangiopericytoma (0/1), or vessel endothelial antigen, clone PAL-E, (hybrid-