00 22-2 02X/84/8~0 3- 0276$02.00/0 THE JOUHNAL OF INVESTIGATIVE DERMATOLOGY, 83:276- 280, 1984 Vol. 83, No. 4 Copyright © 1984 by The Williams & Wilkins Co. Printed in U.S. .4 . Basement Membrane Laminin and Type IV Collagen in Various Benign and Malignant Adnexal Tumors of the Skin: An Immunohistochemical Study MATTI KALLIOINEN, M.D., HELENA AUTIO-HARMAINEN, M.D. KAI DAMMERT, M.D., JUHA RISTELI, M.D., AND LEILA RISTELI, M.D. Departments of Pathology (MK, HA -H, KD) and Clinical Chemistry (JR), and Collagen Research Unit, Department of Medical Biochemistry (JR, LR), University of Oulu, Oulu, Finland Thirty benign and seven malignant adnexal tumors of TABLE I. Basement membranes in adnexal tumors of the skin the skin and one lymph node metastasis were stained for Basement Number Numbers laminin and type IV collagen with rabbit antibodies Origin and membranes of against the human basement membrane (BM) proteins diugnosis (continuous/ of cases leukocytes using the immunoperoxidase technique. Fifteen of the discontinuous) benign sweat gland, sebaceous gland, and hair follicle Benign tumors tumors showed a continuous and distinct BM around the Sweat gland 14 tumor aggregates. The cylindromas and eccrine spira­ Apocrine hidrocystoma 1 1/- denomas seemed to produce excessive amounts of BM Nodular hidradenoma 2 2/- material, part of which was seen as amorphic patches Papillary hidradenoma 2 2/- within the tumor cell clusters, whereas the trichofol­ Eccrine hidradenoma 2 2/- Cylindroma liculomas, trichoepitheliomas, and pilomatrixomas 2 2/- Syringoma 5 -/5 showed an absence of BM from many areas. In syrin­ Hair follicle 14 gomas, in addition to the tubular structures surrounded Trichofolliculoma 3 2/1 by a continuous BM, undifferentiated cell nests contain­ Trichoepithelioma 4 - /4 ing granular BM material were present. They probably Trichilemmoma 2 2/- represent primitive structures obtaining during early Pilomatrixoma 5 -/5 + development into tubules. The seven malignant tumors Sebaceous gland 2 and the only metastasis studied here all contained small, Sebaceous adenoma 2 2/- narrow strips of BM material extracellularly between Malignant tumors the infiltrating tumor clusters. Only in two cases was Sweat gland 5 Hidradenocarcinoma 3 -/3 ++ faint staining for laminin found within the cells. The Apocrine hidradenocar­ 1 -/1 ++ pepsin pretreatment of the formalin-fixed, paraffin-em­ cinoma bedded samples had most probably degraded the intra­ Nodular hidradenocarci­ 1 -/1 + cytoplasmic BM material in most cases. The BM defects noma were found to be associated with malignancy and low Hair follicle 1 differentiation of the adnexal skin tumors, as reported Malignant pilomatrix­ 1 -/1 ++ previously for other tumor types, but a partial loss of oma BM was also associated with high differentiation in some Sebaceous gland 1 Sebaceous carcinoma 1 -/1 + benign adnexal tumors. Metastasis Lymph node metastasis 1 -/1 of sweat gland carci­ The role of the basement membrane (BM) in tumor differ­ noma entiation and growth has been a subject of intensive research lately. Chemically, BMs consist of both collagenous and non­ collagenous glycoproteins (for reviews, see [1,2]). The scaffold goid antigen, which is found in the BMs of stratified squamous of the lamina den sa layers is formed by type IV collagen, which epithelia (5]. is organized in a netlike fashion [3]. The noncollagenous BM Many benign epithelial tumors have an intact BM that can constituents comprise laminin (a glycoprotein of very high be visualized with antibodies to type IV collagen and laminin molecular weight), entactin, a heparan sulfate containing pro­ whereas the BM in their malignant, invasive counterparts is teoglycan, and, at least in some BMs, fibronectin [1,2]. These either thin and discontinuous (6] or completely absent (6,7]. components are probably mainly located in the lamina Iucida The tumor most systematically studied so far is breast carci· layer although all the BM constituents have also been reported noma. No extracellular BM structures can be seen in its me· to be integrated into a common structure [4). In addition to tastases [6,8], whereas intracytoplasmic staining both for lam­ these constituents common to all BMs, some BM structures of inin [6,8] and for type IV collagen [9] has been found. Some limited tissue distribution are known, e.g., the bullous pemphi- benign tumors of mesenchymal origin also produce laminin, and demonstration of this protein may be helpful in distinguish· ing among various types of soft tissue tumors [10]. Manuscript received January 26, 1984; accepted for publication April 26, 1984. Several studies have described the presence of a BM around Supported in part by the Medical Research Council of the Academy basal cell carcinomas of the skin [11 - 15]. This BM is continu­ of Finland. ous and distinct in nonfibrosing tumors, but indistinct or Reprint requests to: Matti Kallioinen, M.D., Department of Pathol­ discontinuous in fibrosing basocellular carcinomas [15]. There ogy, University of Oulu, Kajaanintie 52 D, SF-90220 Oulu 22, Finland. also seems to be a selective Jack of the bullous pemphigoid Abbreviations: antigen in the BM around basal cell carcinomas that distin· BM: basement membrane guishes it from other types of skin tumor [11]. In contrast to I 276 Oct. 1984 BASEMENT MEMBRANE PROTEINS IN ADNEXAL TUMORS 277 t he basocellular carcinomas, there is only limited information available about the BM in adnexal tumors of the skin. Various benign adnexal tumors are quite common findings in derma­ topathology, whereas their malignant counterparts are rare. It is not always easy, however, to tell which adnexal structure the tumor is differentiated toward or which structure it originates from. We have now investigated 37 adnexal tumors with various degrees of differentiation, 30 benign, 7 malignant, with llymph node m etastasis using antibodies to human type IV collagen and laminin and have attempted to find correlations between the immunohistologic findings and the tumor type or the changes that take place in malignancy. MATERIALS AND METHODS Biopsy samples from 30 benign and 7 malignant adnexal tumors, with 1 lymph node metastasis, were selected from the fi les of the Departm ent of Pathology, University of Oulu, and the Pathology Laboratory o f the Cancer F oundation of Oulu, fix ed in 10% formalin and embedded in paraffin. The sections, 5 I'm in thickness were stained with h e matoxylin and eosin, and the tumors classified into sweat gland, sebaceous g land, and hair follicle types (Table I). The number of leukocytes within and around t he tumors was estimated on a scale of negligible (-), moderate(+ ), or abundant(++). T he 7-S collagen domain of type IV collagen was purified from human kidney [1 6] and the fragment P1 of laminin from human placenta [ 17], as described previously, and antisera to these proteins were r aised in rabbits. The antibodies were purified by immunoabsorp- FIG 2. Immunoperoxidase staining for the 7-S domain of type 1 collagen in an eccrine spiradenoma. The t umor clusters are surrounded by a continuous BM (arrows). Amorphic BM material and capillaries (arrowheads) are seen within t hem (X 107). tion on the relevant antige n coupled to Sepharose 4B. The laminin P1 antibodies were cross-absorbed with 7-S coll agen and vice versa. There was no cross-reaction between the two antibodies in t he radioimmu­ noassay. The sections for immunohistochemical staining were deparaffinized and treated with 0.4% pepsin (Sigma Chemical Co., St. Louis, Missouri) to enhance the availability of t he a ntigenic determinants [18,19]. T he sectiOns were exposed to a 0.1 % solut ion of hydrogen peroxide in absolute met hanol to inactivate t he e ndogenous peroxidases, and t hen stained with anti-laminin .P1 (20-40 ,ug/ ml) or anti-7-S collagen (20- 100 ~g/ ml) usmg the perox1dase-ant iperoxidase procedure (20]. Normal rabb1t serum and phosphate-buffered saline were used instead of the primary antibody for control stainings. The BMs were analyzed and classified as follows: (1) continuous, when the BM was seen to envelop every t umor cell aggregate in a di stinct, continuous line without disruptions, and (2) discontinuous, when t he BM around some of the tumor cell aggregates was disrupted or not seen at all. RESULTS All the 37 samples of adnexal tumors of the skin were positive for both the laminin Pl and the 7-S collagen antigen, but showed variations in the integrity of the BM around the tumor cell aggregates. The detailed results are presented in Table I. The staining of the extracellular BMs for both antigens was FIG 1. Jmmunoperoxidase staining for the laminin fragment Pl and identical in each case. The normal epidermal and capillary BMs in a cylindroma. The (inset) for the 7-S domain of type IV collagen stained for laminin Pl and 7-S collagen in every sample, es around t he tumor clusters, beneath the epider­ were basement membran mal rabbit serum and phos­ mis, and around t he dermal capilla ries are continuous and distinct. and the control stainings with nor Abu ndant amorphic BM materi al i s seen within some tumor clusters phate-buffered saline were all negative. (arrows and inset ) (x 44; inset x 90). Fifteen of the 30 benign tumors showed a distinct continuous 278 KALLIOINEN ET AL Vol. 83, No.4 BM around the tumor cell clusters. These included all the cases of apocrine hidrocystoma, nodular and papillary hidradenoma, ecc rine spiradenoma, cylindroma, trichilemmoma, and seba­ ceous adenoma (Table I). The 2 cylindromas showed an unu­ sually thick, homogeneously stained BM around the tumor cell nests and granular or amorphic BM material at the periphery or within the tumor clusters (Fig 1). This material stained equally with anti-7-S collagen and anti-laminin P1 antibodies.