Histol Histopath (1992) 7: 575-582 Histology and Histopathology

Dermal . Expression of intermediate filaments, epithelial and neuroectodermal antigens

U. Wollina, D. Rülke and H. Schaarschmidt Department of Dermatology at the Friedrich-Schiller-University of Jena, Jena, Germany

Summary. We report on irnmunohistochemical staining histochemistry provide additional information? pattems in so-called apocrine tumors of skin with special The dermal cylindroma has light and electron emphasis on the dermal cylindroma. The results microscopic features of an apocrine secretion were compared with apocrine tubular , (Hashimoto et al., 1987; Massa and Medenica, 1987; syringocystadenoma papilliferum and the normal eccrine Lever and Schaumburg-Lever, 1990). On the other hand, sweat . some recent investigators suggest an eccrine A relationship of dermal cylindroma to the apocrine differentiation pathway based upon immuno- gland is suggested by expression of lysozyme and histochemistry of keratins and epithelial antigens like alphal-antichymotrypsin. The tumor shares keratin, CEA (Cotton and Braye, 1984). To possibly resolve this epithelial membrane antigen (EMA) and EGF-receptor controversy, we have investigated dermal expresssion with eccrine and apocrine . The and other apocrine tumors by immunohistochemistry presence of intermingled cells with a coexpression of using three groups of antibodies: against intermediate keratin and vimentin argues for a partial myoepithelia- filaments; epithelial antigens; and neuroectodermal like differentiation. Neuroectodermal antigens are antigens. Comparing their staining pattems with those of rnissing. the normal eccrine of skin (Wollina, 1991), Therefore, dermal cylindroma is classified as an we hoped to gain a better insight into the differentiation adnexal tumor of skin with a variable rate of cells of of dermal cylindromas. apocrine secretory, myoepithelial and undifferentiated phenotypes. Materials and methods

Key words: Apocrine tumors, Immunohistology, Coexpression Samples of human skin tumors were obtained from the tissue bank of the Department of Dermatology, lntroduction University of Jena. These included 12 dermal cylindromas from five patients. In one case, snap-frozen Current classification of adnexal tumors of skin tissue stored in liquid nitrogen was available. It was differentiates eccrine, apocrine and pilosebaceous fixed in acetone for 10 min at 4" C. In al1 other cases, the varieties. An outstanding matter of debate, however, is tumors were fixed in formalin and routinely processed. how one can distinguish between eccrine and apocrine Additionally, syringocystadenoma papilliferum (n=2) glandular tumors. The question is of practica1 impact and apocrine tubular adenoma (n=l) were studied. since there are severa1 tumor entities with apocrine or eccrine differentiation: hydrocystoma or , Antibodies syringocystadenoma papilliferum, basal cell epithelioma with glandular differentiation (Hashimoto et al., 1987). Primary antibodies used in this study are sumrnarized Even the morphological finding of secretion by in Table 1. Rabbit antimouse globulin (working dilution decapitation seems not to be an absolute criterium 1:50), mouse monoclonal alkaline phosphatase- excluding eccrine differentiation. Can immuno- antialkaline phosphatase complex (APAAP; 1: 1000), peroxidase-labelled swine antirabbit and rabbit Orrpflnt requests to: Dr. Uwe Wollina, Deparbnent of Dematology at the antimouse immunoglobulins (1:30) were purchased Friednch-Schiller, University of Jena, 0690OJena, Germany from Dakopatts, Amsterdam, Netherlands. Goat Dermal cylindroma

Table 1. List of primafy antibodies. Antibodies with an asterisk (*) were used only on frozen sections.

Antlbody Origln Characterlzation and worklng dilutlon

1. Antibodies against intermediate filaments

AHK Dakopatts rabbit wide spectrum antikeratin, 1:100 RPN 1161' Amersham Buchler lgG2a broad spectrum antikeratin, 1:10 RKSE 60' Euro-Diagnostics lgGl against keratin #lo, 1:100 K 8.12' BieYeda IgGl against keratins #13/16, 1:100 Cam 5.2 Becton-Dickinson lgG2a against keratins #8/18/19, 1:2 CK 2' Boehringer lgGl against keratin #18, 1:5 PVIM Dakopatts rabbit antivirnentin, 1:50 VIM 9 (1) Monocan lgGl against virnentin, 1:10 NE 14' Boehringer IgGl against neurofilarnent, 1:2 G-A-5' Biochrorn lgGl against GFAPP, 1:5

2. Antibodies against epithelial antigens and Ki67

Filaggrin Paesel lgGl against (pro-)filaggrin, 1:lo0 EMA Dakopatts lgG2 against epithelial membrane antigen (EMA), 1:50 ACAM Dr. l. Wez, Jena rabbit anticalmodulin, 1:50 CEA Dakopatts rabbit anticarcinoernbryonal antigen (CEA), 1:300 29.11. Sigma lgG1 against EGF-receptor, 1:2000 Ki67 Dakopatts lgGl against Ki67 proliferation antigen, 1:5 Lysozyme Dakopatts rabbit antilysozyrne, 1:400 AACT Dakopatts rabbit antibody to alphal -antichymotrypsin (AACT), 1:500 3. Antibodies against neuroedtodermal antigens

S-100 Dakopatts rabbit anti-S-100, 1:20 NSE' Dakopatts rabbit antibody to neuron-specific enolase (NSE), 1:25 NKIIC-3 Monocan lgGl against melanoma-associated antigen, 1:20 LS 59 Dr. L.M. Jeny lgG2a against neuroglandular antigen, 1:lo0 (CalgaryICanada) antimouse bridging irnrnunoglobulin (1:25) from Tago, Primary antibodies were diluted in PBS BurlingameICA, was used in double staining supplemented with bovine serum albumin, supplemented experiments. with 0.2M glucose and 1.SIUlml glucose oxidase (Boehringer Mannheim, FRG) and incubated for one Sfaining procedures hour. Peroxide oxidase (POX) staining was performed according to Mason and Sammons (1987) with 3-amino- Serial sections were cut at 8 pm from formalin-fixed, 9-ethylcarbazole (AEC), purchased from EGA-Chemie, paraffin-embedded blocks, dewaxed by Histosol Steinheim, FRG. The sections were counterstained with (Merck, Darmstadt, FRG) and rehydrated through Mayer's hemaiun (Merck). decreasing concentrations of ethanol, followed by Frozen sections were processed to 4 pm thick, and washing in deionized water for 10 min. Al1 incubation acetone-fixed specimens stained with POX or APAAP as steps except otherwise noted were performed at room described elsewhere (Mason and Sammons, 1987). temperature. Endogenous peroxidase was inactivated Naphtol-As-phosphate (Sigma) served as coupler. with methanol-acetone (20 min). Subsequently the slides For double-staining on both formalin- and acetone- were rinsed in phosphate-buffered saline (PBS) for 10 fixed sections we combined the APAAP technique with min. Fast Blue BB salt (Sigma) and POX with AEC (Mason Sections were trypsinized (0.1% in distilled water) and Sammons, 1987). TRIS buffer was used instead of for 30 min at 37' C, rinsed in PBS and overlaid with the PBS. The following antibody combinations were appropriate normal swine or rabbit serum (1:3 diluted in performed: PVIM plus Cam 5.2; Vim 9 (1) with AHK; K PBS) for 30 min at room temperature. Trypsinization 8.12 or RPN 1161; AHK and Ki67. In these protocols, was omitted before staining with antibodies against counterstained was omitted. neuron-specific enolase (NSE), calmodulin (ACAM), In controls, primary antibodies were substituted by NKVC-3 and LS59. buffer. No staining was observed. Dermal cylindroma

Flg. 1. Dermal cylindroma; staining with monoclonal antibody RPN 1161 against keratin. Note the different stages of lumen formation. x 250

Results CEA (Fig. 5). The amorphous luminal content was strongly reactive for CEA and moderately reactive for Dermal cylindroma (Table 2) AMA or S-100 (Fig. 5c). Vimentin was not demonstrated. Occasionally, scattered LS 59-positive In dermal cylindromas, the majority of cells were cells were found within the . The apocrine non-reactive with antibodies against keratins. Cell tubular adenoma was lysozyme-positive. forming duct-like structures or squarnous whorls and scattered single, in part ring-like ceiis were labelled with Discussion AHK, Cam 5.2, RPN 1161, and CK2 (Fig. 1). This particular cell type also expressed a number of epithelial Irnrnunohistochemistry comes to the forefkont in the antigens like EMA, EGF-receptor, lysozyme and alphal- diagnosis of adnexai cutaneous neoplasms. The problem antichymotrypsin (Fig. 2). of their classification is exemplified by dermal Scattered cells of solid areas were vimentin-positive cylindroma, which was morphologically defined as (Fig. 3). Double-staining revealed that some keratin- apocrine, whereas histochemistry suggested an eccrine positive cells simultaneously expressed vimentin (Fig. phenotype (Cotton and Braye, 1984; Hashimoto et al., 4). There was no additional evidence for coexpression of 1987; Massa and Medenica, 1987; Lever and intermediate filaments. The tumors were completely Schaumburg-Lever, 1990). negative for neurofilaments and glial fibrillary acidic Dermai cylindroma is composed of solid and lumen- protein (GFAP). Both filaggrin, and high-molecular forming cells. The lumen formation shows a variable keratins were missing. degree ranging from ring-like single cells, resembling None of the neuroectodermal antigens (NSE, NKVC- embryologic pre-luminai cells of the dermal sweat duct 3, LS 59) were observed. Occasionally, ACAM (Hashimoto et al., 1966), to tal1 columnar lumina disclosed a nucleolar or perinucleolar staining in tumor showing decapitation secretion like the apocrine cell clusters. Within solid tumor masses scattered Ki67- secretory segment (Hashimoto et al., 1987). From an positive cells were seen in the frozen specimen. As immunohistochemicai point of view, these cells are the demonstrated by double-staining, the Ki67-positive, most mature expressing a number of different antigens proliferating cells expressed keratin. but missing Ki67-labelling. Simple-type keratins (# 8, 18, 19) and epithelial Syringocystadenoma papilliferum and apocrine tubular antigens of both secretory coils and inner duct cells of adenoma of skin eccrine glands, like EMA and EGF-receptor have been detected in the lumen-lining epithelium of cylindromas Most of the columnar, lumen-lining epithelium was (Cotton and Braye, 1984; Nanney et d., 1984; Noda et strongly positive for Cam 5.2, but only a minority for al., 1987, 1988). Almost al1 of these antigens are present Dermal cylindroma

Flg. 2. Derrnal cylindroma. (a) Staining with monodonal antibody 29.1 1 against EGF-receptor. Decoration of upreluminaln, ring- like cells. x 250. @) Predominant luminal staining with a polyclonal antibody against alphal-anti- chymotrypsin. x 100 in normal eccrine sweat glands (Cotton, 1986; Iki, 1990; the normal eccrine sweat glands (Ezoe and Katsumata, Wollina et al., 1990, 1991; Wollina, 1991). Definitive 1990; Wollina, 1991) as well as in benign eccrine tumors conclusions could not be drawn from the keratins, CEA of skin (Katsumata and Ezoe, 1990). Adnexal tumors of and EMA, since they have also been detected in apocrine the apocrine type express lysozyme (Katsumata and glands (Penneys, 1984, Noda et al., 1987, 1988). Ezoe, 1990). We also observed lysozyme-positive Otherwise, a secretory phenotype is a reasonable epithelial cells in apocrine tubular adenoma. deduction from these findings. Among the dermal cylindromas, there was evidence On 'the other hand, lysozyme and alphal- for coexpression of vimentin and keratin in a minority of antichymotrypsin found in cylindromas were absent in cells localized within solid tumor masses. Some of them Dermal cylindroma

FIg. 3. Dennal cylindroma; staining with polyclonal antivimentin. Scanered cells within the didtumor mass are decorated. x 250

Tabk 2. Overview of immunohistodiemicai staiiing pattems of demal cylindroma and he normal (according to Woilina, 1991).

AHK RPN 1161 RKSE 80 K 8.12 Cam 5.2 + (PO PVlM + (PID) Vim 9(1) + (very few P/few D) NE 14

2. Epithelkil an-8 and Ki67

Filaggtin EMA + (PO ACAM - (most S) CEA 29.1 1 + KB7 + (few S)' Lysozyme + (pm) AACT + (PO

S100 NSE NKVC-3 LS59

Fig. 4. Demal cylindroma; double-staining with pdyclonal antikeratin *: On frozen dons; P: preluminal as; D: duct-linlng cdelEs; S: solid and monodonal Vim 9 (1) demonstrates cells coexpressing both keratin areas of ~indromas;A: acroayringium; ID: imer duct epithelium; OD: and virnentin . x 250 outer duct epithellum; SC:secretoly oolls; M: myúepithelial cells. Dermal cylindroma

Fig. 5. lmmunostaining of apocnne tumors. Synngocystadenoma papillife~m(a) and apocrine tubular adenoma (b - c). (a, b): Demonstration of simple-type keratins with antibody Cam 5.2 in the lurnen-lining epithelium; a) APAAP, x 100; b) peroxidase technique, x 250. (c) lmrnunostaining for CEA; peroxidase technique, x 250 Dermal cylindroma

showed a ring-like appearance. lnterestingly enough, References the coexpression of both intermediate filaments by myoepithelial cells of normal eccrine sweat Conon D.W.K. (1986). lmmunohistochernical staining of normal sweat glands was described recently (Wollina, 1991). glands. Br. J. Dematol. 114,441-449. Myoepithelial cells of severa1 other exocrine glands Cotton D.W.K. and Braye S.G. (1984). Demal cylindromas originate are also capable of coexpressing vimentin and from the eccrine sweat gland. Br. J. Dermatol. 111,53-61. keratin ~(McGuireet al., 1989). Nanney et al. Ezoe K. and Katsumata M. (1990). lmmunohistochemical localization of (1984) reported a strong immunoreactivity lysozyme in human apocrine glands. J. Dermatol. 17, 159-163. of myoepithelial cells but not secretory segments Graham Jr. R.C., Lundholm U. and Karnovsky M.J. (1965). with antibodies against EGF-receptor. Expression Cytochemical demonstration of peroxidase activity with 3-amino-9- of EGF-receptor by cylindromas provides additional ethylcarbazole. J. Histochem. Cytochern. 14, 150-152. support for the presence of a myoepithelial phenotype. Hashimoto K., Gross B.O. and Lever W.F. (1966). The ultrastructure of In conclusion, the dermal cylindroma has much human embryo skin. II. The formation of the intradermal portion of in common with apocrine secretory segments. Cells the eccrine sweat duct and of the secretory segment during the first of a myoepithelial phenotype can be found within half of embryonic life. J. Invest. Dermatol. 46, 513-529. solid areas and among ring-like cells with co- Hashimoto K., Mehregan A.H. and Kumakiri M. (1987). Tumors of skin expression of keratin and vimentin. And a appendages. StonehamiMA. Butterworth Publishers second practica1 conclusion can be drawn: Iki M. (1990). lmmunohistochemical studies on eccrine sweat apparatus CEA, EMA and simple-type keratins are not neoplasms. Part 1. An irnmunohistochemical study on the eccrine reliable «markers» that distinguish eccrine from sweat apparatus. Skin Res. 32, 345-352. apocrine sweat glands, as assumed earlier (Cotton, Katsumata M. and Ezoe K. (1990). lmmunohistochernical study of 1986). lysozyme in various benign sweat apparatus tumors. J. Dermatol. 17, 307-31 1. Acknowledgements. We are very grateful to Prof. Gustav Lever W.F. and Schaumburg-Lever G. (1990). Histopathology of the Mahrle, Department of Dermatology, University of Kbln, who skin. J.B. Lippincott. Philadelphia. Toronto. provided excellent working facilities in his laboratory for a part Mason D.Y. and Samrnons R. (1978). Alkaline phosphatase and of the investigations. The technical assistance of Miss peroxidase for double immunoenzymatic labelling of cellular Sabine Feldrappe (Jena) is highly appreciated. The work was constituents. J. Clin. Pathol. 31, 454-460. supported by a grant (UW) from the Paul-Gersson-Unna-Stiftung, Masa M.C. and Medenica M. (1987). Cutaneous adnexal tumors and Düsseldorf. . Part. II - Tumors with apocrine and eccrine glandular differentiation and miscellaneous cutaneous cysts. Pathol. Annu. 22, Dermal cylindroma

225-276. 291. McGuire L.J., Ng J.P.W. and Lee J.C.K. (1988). Coexpression of Penneys N.S. (1984). Irnrnunohistochemistty of adnexal neoplasms. J. cytokeratin and vimentin. Appl. Pathol. 7, 73-84. Cutan. Pathol. 11,357-364. Nanney L.B., Magid M., Stoscheck C.M. and King Jr. L.E. (1984). Wollina U. (1991). Human eccrine sweat gland. Expression of Comparison of epidermal growth factor binding and receptor neuroglandular antigens and coexpression of intermediate filaments. distribution in normal human epidemis and epidemal appendages. Histol. Histopath. 6, 191-198. J. Invest. Dermatol. 83,385-393. Wollina U, Schaarschmidt H. and Knopf B. (1990). lmmunolocalization Noda Y., Horike H., Watanabe Y., Mori M. and Tsujimura T. (1987). of cytokeratins in hurnan eccrine sweat glands. Acta Histochem. 88. lrnmunohistochemical identification of epithelial membrane antigen 125-133. in sweat gland tumours by the use of monoclonal antibodies. Pathol. Wollina U, Henkel U., Schaarschmidt H., Knopf B. and Hipler C. (1991). Res. Pract. 182,797-804. Human eccrine sweat glands - a histochemical approach. Anat. Anz. Noda Y., Kumasa S., Higoshiyama S. and Mori M. (1988). 173. 155-159. lmmunolocalization of keratin proteins in sweat gland tumours by the use of monoclonal antibody. Pathol. Res. Pract. 183, 284- Accepted May 2,1992