Basement membranes in neoplasia.

Basement Membranes in Neoplasia

F. T. BOSMAN· M. G. HAVENITH R. VISSER· J. P. M. CLEUTJENS

With 40 Figures and 1 Table

~! SEMPER

~

GUSTAV FISCHER VERLAG· STUTTGART· NEW YORK· 1992

FRED T. BOSMAN':', M.D., Ph.D. Professor of Pathology MICHAEL G. HAVENITH, M.D., Ph.D. ROB VISSER, M.D. JACK P. M. CLEUTJENS, Ph.D. Department of Pathology University of Limburg, Faculty of Medicine P.O. Box 616, NL-6200 MD Maastricht (The Netherlands) ':. Present address: Department of Pathology Erasmus University Rotterdam P.O. Box 1738, NL-3000 DR Rotterdam (The Netherlands)

Acknowledgements The investigations reported in this volume were supported by a grant from the Netherlands Cancer Foundation (grant no. RUL 85-2). The authors gratefully acknowledge the expert secre tarial assistance of Claire Bollen. Dr. P. M. Frederik prepared figures 5 and 7.

Die Deutsche Bibliothek - CIP-Einheitsaufnahme Basement membranes in neoplasia I F. T. Bosman ... - Stuttgart; New York; Jena : G. Fischer, 1992 (Progress in histochemistry and cytochemistry; Vol. 24, No.4) ISBN 3-437-11435-2 (Stuttgart ... ) ISBN 1-56081-347-4 (New York ... ) NE: Bosman, Fred T.; GT Library of Congress Card-No. 88-20469

Published jointly by: Gustav Fischer Verlag/VCH Publishers 220 East 23rd Street, Suite 909, New York, New York 10010 Gustav Fischer Verlag Wollgrasweg 49, D-7000 Stuttgart 70 (Hohenheim) FRG © Gustav Fischer Verlag· Stuttgart· Jena . New York· 1992 AIle Rechte vorbehalten Gesamtherstellung: Laupp & Gobel, Nehren/Tiibingen Printed in Germany

Contents 1 2 2.1 2.2 2.3 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5 2.3.6 2.3.7 2.3.8 2.4 3 3.1 3.2 3.3 3.4 ~

4.1 4.2 4.2.1 4.2.2 4.2.3 5 5.1 5.1.1 5.1.2 5.1.3 5.2 5.2.1 5.2.2 5.2.3 5.2.4 5.2.5

Introduction . . . . . . . . . . . . . . . . . . . . . . . 1 Structure and composition of the basement membrane 2 Basement membrane distribution. . 2 Basement membrane ultrastructure 4 Basement membrane composition 7 Type IV collagen 9 Laminin...... 10 Merosin...... 11 Nidogen (entactin) 11 Heparan sulphate proteoglycan . 11 Type VII collagen . . . . . . . . 12 Bullous pemphigoid antigens . . 12 Basement membrane associated components 13 Basement membrane assembly . . . . . . . . 14 Interaction of neoplastic cells with basement membranes 15 Basement membrane degradation in invasive growth 15 Basement membrane deposition by neoplastic cells . . . 18 Cellular attachment to basement membranes 23 Modulation of cellular growth and development by basement membrane components . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Basement membrane histochemistry: Methodological aspects 27 Conventional histochemistry and electron microscopy 27 Immunohistochemistry. . . . 28 Tissue processing procedures . 28 Antibody specificity 29 Choice of label 30 Basement membrane immunohistochemistry as a tool in the histopatho logical diagnosis of cancer. . . . . . . . . . . . 31 General principles . . . . . . . . . . . . . . . . . . . . . 31 The basement membrane and invasive growth . . . . . . 31 Basement membranes and differentiation in carcinomas. 33 Basement membranes and classification of soft tissue sarcomas . 33 Skin. . . . . . . . . . . . . . . 35 Benign and preneoplastic lesions . 36 Epidermoid carcinoma . 36 Basal cell epithelioma . . . . 37 Nevomelanocytic lesions . . 38 Cutaneous adnexal tumours 40

VI . Contents

5.3 5.3.1 5.3.2 5.3.3 5.4 5.4.1 5.4.2 5.5 5.5.1 5.5.2 5.5.3 5.5.4 5.5.5

5.6 5.7 5.7.1 5.7.2 5.7.3 5.7.4

5.8 5.9 5.9.1

5.9.2 6 6.1 6.1.1 6.1.2 6.1.3 6.1.4 6.2 7 8

The breast . The normal breast . . . . . . . . . Benign proliferative breast lesions Breast carcinoma . . . . . The respiratory tract . Upper respiratory tract tumors . Lower respiratory tract The digestive tract . . . . . Salivary glands. . . . . . . Oral tissues and esophagus Stomach, small and large bowel . The pancreas. . . . . . . The hepatobiliary system Endocrine system . . Genito-urinary tract The kidneys . . . . Ureter and bladder . Male genital tract . . The female genital tract Nervous system . . . Mesenchymal tissues Bones and joints . . . Soft tissues . . . . . . Concluding remarks Diagnostic use of basement membrane immunohistochemistry . General tissue architecture Invasive growth Prognosis . Differentiation . Basement membranes and the pathobiology of neoplasms References . . Subject index .

40 40 41 42 43

44 44 47 47 48 49

52 53 54

56 56 58 59 62

65 68 68 68 72 72 72 72 73

74 74 75 90

1 Introduction Neoplastic growth is one of the most intriguing pathobiological phenomena. Un ravelling the reasons why the growth of neoplastic cells is abnormally regulated is as much a challenge as clarifying the growth regulation of normal cells. How neoplastic cells, when they acquire the potential for invasive growth, leave their original tissue compartment and gain access to adjacent compartments is still a matter of intensive investigation. How malignant cells that have entered the circulation are able to survive, lodge and recolonize in a metastatic site remains largely an open question despite massive research efforts. From a biological point of view, neoplasia is an intriguing phenomenon because it exemplifies how the study of a pathological principle may lead to fundamental insight into the natural regulatory mechanism of cell growth. From a medical point of view neoplasia is equally important. Neoplasia is the second most relevant cause of death (after cardiovascular diseases) in the Western world and massive efforts are being di rected towards early diagnosis and adequate treatment of cancer which might improve the grave prognosis of many types of cancer. In the area of cancer cell biology, attention has been focussed largely on the charac teristics of cancer cells as such. Much less attention has been paid to interactions between cancer cells and between cancer cells and host tissue elements, though these may be important determinants with regard to the behavior of the neoplasm as a whole. The reaction of the host in terms of the immune response elicited by neoplasia has come in for extensive study. Non-immunological host responses have, however, been almost ~ntirely neglected. The interactions between neoplastic cells and the extracellular matrix fall into this category. It is obvious that when carcinoma cells proliferate, stromal elements contain ing at least fibroblasts and capillaries will have to proliferate in a rather concerted action if a viable tumor is to be yielded. How tumor cells stimulte the development of a tumor stroma and, conversely, how stromal elements influence the growth and differentation of neoplastic cells have long been entirely neglected questions. In recent years, however, increasing attention has been paid to the interactions between the tumor stroma, largely consisting of extracellular matrix, and the neoplastic cells. A classical concept in cancer biology has been the penetration of invasively growing, malignant tumor cells via the epithelial basement membrane into the connective tissue compartment. It has become dear, however, that this concept is an extreme oversimplification. Tumor cells may deposit basement membrane material and, at the same time, may elaborate basement membrane-specific proteases. This implies that the presence of a basement membrane mrrounding neoplastic cells is largely determined by the balance between synthesis and iegradation. It has also become clear that stromal elements play an active role in basement membrane deposition. In this respect the so-called desmoplastic reaction, i.e. the deposition of rather dense collagen containing numerous myofibroblasts around a

2 . F. T. Bosman et al.

neoplasm, is a characteristic phenomenon and should be regarded as an important aspect of the host response to neoplasia (VAN DEN HOOFF et al. 1983). The theoretical importance of these phenomena may be self-evident but they also have a significant practical importance. In diagnostic pathology it is frequently difficult to distinguish between non-invasive pre- or potentially malignant lesions, on the one hand, and the initial phase of invasive growth in malignancies, on the other. In this area, studies concerning the interactions between neoplastic cells and the basement mem brane may be fruitful. A simple working concept would be that in a benign lesion basement membranes are architecturally and structurally intact whereas in malignant lesions they show interruptions and structural abnormalities. In this volume the interactions between neoplastic cells and the extracellular matrix are reviewed with particular emphasis on the basement membrane. Normal anatomy and chemical composition of the basement membrane are discussed and the most im portant characteristics of the molecular components of this structure are reviewed in Chapter 2. In Chapter 3, the fundamental aspects of the interactions between neoplastic cells and the basement membrane are taken up. Particular attention is paid to how neoplastic cells deposit as well as degrade basement membranes and how basement membrane components, for their part, modulate growth and differentiation of the adjacent neoplastic cells. In Chapter 4, immunohistochemical techniques for basement membrane staining are reviewed. In Chapter 5, the use of immunohistochemical detec tion of basement membrane components in diagnostic pathology is discussed. Particular attention is paid to the distinction between benign and malignant lesions and the corre lations between basement membrane desposition, tumor differentiation and tumor be havior.. In Chapter 6, conclusions are drawn and future developments speculated upon.

2 Structure and composition of the basement membrane 2.1 Basement membrane distribution The basement membrane is a specialized compartment of the extracellular matrix. Basement membranes are deposited as sheet-like structures in the close vicinity of the adherent cell. These structures may be deposited in a polarized fashion at the basal side of such cells as epithelia and endothelia (Fig. 1, 2). Other cells - such as adipocytes (Fig. 15), myocytes and Schwann cells - are completely surrounded by a basement membrane (VRACKO 1982). Myofibroblasts display at their surface irregular and discon tinuous deposits of basement membrane material (Fig. 3). Occasionally, epithelia do not rest upon a structurally recognizable basement membrane. This situation is encoun tered, for instance, in the central cornea (CLEUTJENS et al. 1990) (Fig. 4) where the epithelium lacks a lamina densa and rests directly upon Bowman's membrane. The epithelial lining of the eye lens is demarcated by a basement membrane on the lens

Basement membranes in neoplasia . 3

Fig. 1. Basement membrane distribution in the human epidermis, visualized by immunohis :ochemical staining for type IV collagen. Basement membranes occur adjacent to the epidermis :E), vascular structures (V) and sweat glands (5). - Immunoperoxidase (x 60).

mrface and not on the interface between epithelium and lens stroma. A special situation ~xists also in the placental membranes. Here a distinct basement membrane underlies :he amnion epithelium, but individual decidual chorionic cells are surrounded by a ;omewhat irregular basement membrane. A striking example of basement membrane :lynamics is found in endometrial stroma where in the luteal phase of the menstrual ;ycle decidualized endometrial stromal cells are surrounded by a basement membrane :CHARPIN et al. 1985; WEWER et al. 1985).


Fig. 2. Human cornea. Epithelial cell in the corneal periphery resting upon a continous basement membrane. E corneal epithelium, LL lamina lucida, LD lamina densa, AF anchoring fibers. Scale bar 0.1 !J.m.

2.2 Basement membrane structure Ultrastructurally, basement membranes appear as a three layered structure (MAR TINEZ-HERNANDEZ and AMENTA 1983; ABRAHAMSON 1986; GORSTEIN 1988). Adjacent to the plasma membrane of the adherent cell an electron lucent layer can be disting uished: the lamina rara or lamina lucida (Fig. 2). In this lamina very delicate filaments occur which traverse this layer perpendicularly to the plasma membrane of the adherent cell (EADY 1988). The lamina densa is located adjacent to the lamina lucida. At high magnification this lamina appears as a network of cords. The subbasal lamina or lamina fibroreticularis is found on the stromal side of the basement membrane. This lamina contains fibrils attaching the basement membrane to the underlying stroma. These fibrils include anchoring fibrils, elastic microfibrils and fine interstitial fibrils, which probably represent reticulin (INOUE and LEBLOND 1988).


:;ig. 3. Detail of umbilical cord myofibroblast. Note peripheral myofilaments and discontinuous deposits of basement membrane like material (BM). The cell is surrounded by :xtracellular matrix displaying cross sectioned collagen fibrils. - Scale bar 0.1 Jlm. ~xtracellular

Epithelial cells are attached to the basement membrane by hemidesmosomes. These :ontain an intracellular attachment plaque, which is in contact with the plasma mem )rane and with intracytoplasmic actin and keratin tonofilaments; they also contain an :xtracellular subbasal dense plate which is located in the basement membrane (Fig. 5). Anchoring filaments are more numerous in the hemidesmosomal region than be ween the hemidesmosomes. In contrast, anchoring fibrils occur predominantly at the lemidesmosomal sites (INOUE and LEBLOND 1988;GIPSON et al. 1988; TISDALE et al. 1988). As a result, the anchoring of epithelial cells is mediated by a complex network of ntracytoplasmic filaments, hemidesmosomes, basement membrane components and Lllchoring fibrils. Cell-matrix attachment is also mediated by specific binding sites for


Fig. 4. Human cornea. Epithelial cell (E) in the center of the cornea rests on stroma (S) without a continuous basement membrane at the interface. - Scale bar 0.1 !J.m.

extracellular matrix components on the cell surface (RUOSLAHTI and PIERSCHBACHER 1986; AUMAILLEY et al. 1987). Although at first sight basement membranes seem to be rather homogeneous, close scrutiny of these structures has shown that a remarkable degree of heterogeneity exists (BOSMAN et al. 1989). Basement membranes differ in continuity, in their laminar struc ture and in the thickness of the individual layers. Although in most situations basement membranes appears as a continuous layer, patches of electrondense material occur around myofibroblasts (Fig. 3); the former exhibit the components characteristic for basement membranes (LIPPER et al. 1980; SEEMAYER et al. 1980; 1981). Likewise, in the liver sinusoids, Disse's space is delineated by small, discontinuous patches of type IV collagen (MARTINEZ-HERNANDEZ 1984a, b). Furthermore, in the small intestines fenest rations have been observed especially over Peyer's patches (KOMURO 1985; MCCLUG AGE et al. 1986). In some situations the basement membrane laminar structure displays peculiar characteristics (INOUE 1989). A lamina fibroreticularis, for example, can only be found

Basement membranes in neoplasia

0

7

~ig. 5. Schematic representation of the interaction between an epithelial cell and the basement nembrane. The intercellular attachment plaque, which is in contact with cytoplasmic (actin and :ytokeratin) filaments, connects with the extracellular subbasal dense plate in the basement mem >rane. The basement membrane is anchored in the interstitial stroma through type IV collagen :ontaining anchoring fibers.

n the basement membranes of the epidermis and epidermal adnexa, glandular epi helium of breast and prostate, squamous epithelium of the tongue, esophagus and oagina and ciliated epithelia of the trachea and bronchus (SAKAI et al. 1986). In some ites, the basement membrane contains extra layers. This occurs characteristically in the enal glomerular and pulmonary alveolar basement membranes, which appear as a three lyered structure composed of a lamina densa between two laminae lucida (externa and "lterna) (Fig. 6). Moreover, the lamina densa may vary somewhat in thickness. Espe ially at the attachment sites of hemidesmosomes this lamina appears to be somewhat hicker (TISDALE et al. 1988).

~.3

Basement membrane composition

Because of a multitude of covalent and non-covalent interactions between the mac omolecules which compose this structure, basement membranes are highly insoluble. \.s a consequence, it appeared rather difficult to isolate and analyze purified intact omponents. A major step forward in the study of basement membrane composition las come with the propagation of the Engelbreth-Holm-Swarm (EHS) tumor in lathy itic mice (ORKIN et al. 1977). This tumor produces massive amounts of basement


Fig. 6. Glomerular basement membrane. This appears as a trilaminar structure between en dothelium (EN) and epithelium (EP). - Scale bar 0.1 Ilm.

membrane material which, in situations of defective cross linking, can be easily dissol ved and analyzed in a relatively unaltered form. These studies have led to the identifica tion of type IV collagen, laminin, heparan sulphate proteoglycan and entactin/nidogen as intrinsic components in almost all basement membranes. A large number of other components have been identified, most of which do not occur exclusively in the base ment membrane. Both the components and their chemical and functional characteristics are listed in Table 1.

Basement membranes in neoplasia . 9 Table 1. Basement membrane components and their characteristics. Component

Molecular characteristics

Function

type IV collagen

triple helix; 4 U chains (1,2,3,4); assembles into a network; col lagenous and globular domains

BM scaffold; interaction with laminin, fibronectin and proteo glycans

laminin

Mw 900 kD; heterotrimer of A, B1 and B2 chains; characteristic cross shape

adheres to type IV collagen; mediates cell binding; interacts with nidogen

nerosm

Mw 80-300 kD, similarity to C terminus of laminin

tissue specific distribution (trophoblast, muscle, Schwann cells); mediates cell adhesion

linear molecule with globular do mains; Mw 145 kD; sequence homology with EGF

interacts with laminin and me diates cell adhesion

leparan sulphate )roteoglycan

protein core; 50-80% heparan sulphate side chains

kationic; regulates filtration of macromolecules

ype VII collagen

170 kD triple helical domain whith 150 kD non helical (C-terminal) domain

association with anchoring fib ers; anchoring of BM to intersti tial stroma

mllous pemphigoid mtigen (BPA)

220 kD, probably a dimer

hemidesmosomal component; mediates cell adhesion

~ntactin

(nidogen)

!.3.1 Type IV collagen Type IV collagen is the basic structural component of the basement membrane. As is true of all :ollagens, type IV collagen has a triple helix structure. The triple helix is composed of a homo- or leterotrimer consisting of four different u chains, of which the UI (IV) and U2 (IV) chains were the irst to be recognized. Most of the type IV collagen molecules have a [UI (IV)]2 [U2 (IV)] composi ion (TIMPL 1989). The genes for these chains have been cloned (SOININEN et al. 1986, 1988; xrOOD et al. 1988). Recently two new chains, the U3 (IV) and u4(IV), were identified (SAUS et al. 988). Type IV collagen differs from the interstitial collagens (type I and III) through having a rather haracteristic multidomain structure (TIMPL et al. 1981). The triplet sequence Gly-X-Y shows requent interruptions, in which X or Y is often proline or hydroxyproline (SCHUPPAN et al. 980). The triple helix is connected at its amino-terminal domain with three other amino-terminal lomains (partly by covalent interactions) and together these form the so-called 7-S domain RISTELI et al. 1980; DIXIT et al. 1984; FURTHMAYER et al. 1985). The globular carboxy-terminal lomain is another cross-linking site, which participates in the formation of a three-dimensional l1atrix. At these domains, two type IV collagen molecules are connected, partly by disulphide

10 . F. T. Bosman et at.

bonds and partly by covalent cross-linking, to form the non-collagenous (NC 1) domain (WEBER et al. 1984; WIESLANDER et al. 1984, 1985). Six of these globular NC-1 end regions assemble to form a hexameric configuration. Interactions along the type IV collagen molecule can also initiate lateral assembly (TSILIBARY and CHARONIS 1986). Within the triple helix another non-collagenous segment can be found, the so-called NC-2 domain. This segment contains triple helical and non triple helical sequences and probably acts as a flexible hinge (TIMPLE and DZIADEK 1986). The insolubility of type IV collagen is due to the formation of large networks which are stabilized by disulphide bridges and non-reducible cross-links (ABRAHAMSON 1982; MARTINEZ-HERNANDEZ and AMENTA 1983; TIMPL and DZIADEK 1986). Type IV collagen is the basic structural compoment of all basement membranes. However, remarkable variations exist in the concentration of this protein in different basement membranes. The glomerular basement membrane, for example, has as much as 45% of its dry weight in the form of type IV collagen, whereas with Reichert's membrane this amounts to only 25% (KEFALIDES et a1. 1979). In the central corneal epithelium type IV collagen appears to be lacking altogether (CLEUTJENS et a1. 1990). 2.3.2 Laminin Laminin is the most abundant glycoprotein in basement membranes. This molecule is structurally important but also biologically active. As type IV collagen, laminin was first isolated in intact form from the EHS tumor (TIMPL et a1. 1979). Subsequently it was also isolated from murine parietal endoderm cells (COOPER et a1. 1981). The lami nin molecule (Mw 850 kD) is a cross shaped structure with one long and three short arms. Three distinct polypeptide chains have been recognized and designated A, Bland B2, these being interconnected by disulphide bonds. Each chain consists of a rod terminating in a complex globular domain, which has been shown to consist of three smaller globules (PAULSSON et a1. 1985) The genes coding for the mouse laminin chains have been cloned (SASAKI et al. 1987; HART et al. 1988) and the aminoacid sequences have been established. More recently, the human laminin genes have also been cloned (OLSEN et al. 1989). The B1 and B2 chains appear to be significantly homologous. The N-terminal domains contain cysteine-rich repeating segments, a structure which is also encountered in the epidermal growth factor, the transforming growth factor ~, coagulation factors and thrombospondin (LAWER and HYNES 1986). Whereas the A chain contains a cell binding RGD sequence, the B1 chains contain a YGSR site of importance in cell spreading and metastasis. It is not likely that isoforms of laminin exist. This could be due to differences in chain structure or chain composition (KLEINMAN et al. 1987; OLSEN et al. 1989). Furthermore, variable reactivity with anti-laminin antibodies after proteolytic digestion has been described (LEE et al. 1988), a fact which also reflects the existence of isoforms. The role of laminin as a structural component of the basement membrane implies that the molecule can interact with other components of the former. First of all laminin interacts with itself, spontaneous interaction occuring under defined conditions be tween globular domains on both the short arms and the long arms. The binding of


laminin to type IV collagen occurs only when the triple helical configuration is intact and occurs on one or two major binding sites along the triple helical domain. On laminin the collagen type IV binding sites reside in the globular domains of both the short (RAa et a1. 1982) and the long arms (LAURIE et a1. 1986). With nidogen (or entactin) laminin forms highly stable complexes. The nidogen binding site resides in the center of the laminin cross (MARTIN and TIMPL 1987). Weak binding of laminin with heparan sulphate proteoglycan occurs via the heparan sulphate chains of the molecule. The significance of this interaction, however, is not clear. 2.3.3 Merosin Recently merosin, a novel basement membrane protein, was detected in basement membranes of trophoblast, Schwann cells and striated muscle (LEIva and ENGVALL 1988). This apparently tissue specific basement membrane protein has been shown to occur in diffe rent tissue forms of 80 and 300 kD. Cloning and sequencing of the gene has shown that the C terminal domain of merosin exhibits about 40% sequence homology with the A-chain of laminin md is thus also similar in subdomain structure. l.3.4 Nidogen (entactin)

Nidogen was originally isolated as an extracellular matrix component in the EHS tumor :TIMPL et a1. 1983). Previously, entactin had been isolated as a protein of similar molecules mass :CARLIN et a1. 1981). Chromatographic studies have shown that these proteins are highly similar )r even identical (PAULSSON et a1. 1986). It appears to be a dumb-bell shaped protein of Mw 150 ill, highly susceptible to proteolytic degradation (TIMPL et a1. 1983; PAULSSON et a1. 1986). R.ecently the complete aminoacid sequence has been derived from sequencing cDNA clones DURKIN et a1. 1988). It was shown that entactin has sequences homologous with the epidermal ;rowth factor and with low density lipoprotein receptors. The high affinity of nidogen for laminin was initially indicated by the fact that ;pecific antibodies induced its coprecipitation with laminin (DZIADEK and TIMPL 1985). ,ubsequent studies have shown that nidogen binds via its globular domains to the center )f the laminin cross. This suggests that the molecule may possess a bridging function in :he basement membrane. U.S Heparan sulphate proteglycan

Glycosaminoglycans, among which heparan sulphate proteglycan (HSPG) is found, ;onstitute an important component of the extracellular matrix (TRELSTAD 1985). HSPG )robably consists of a family of molecules which may differ in their core protein as well lS in the ratio of protein to glycosaminoglycan. As much as 50-80% of the molecule nay consist of heparan sulphate side chains (HASSEL et a1. 1980). Proteoglycans of


different core sizes and different heparan sulphate chain sizes have been isolated. Both a high-density and a low density HSPG have been demonstrated (YURCHENKO and SCHIITNY 1990). HSPG was shown by these authors to occur in the basement mem brane, predominantly in the lamina lucida where it forms clustered aggregates. Others have found HSPG also in the lamina densa (FINE 1985). Strong binding of the protein core with other matrix proteins is a likelihood (PAULSSON et a1. 1985, 1986). HSPG probably plays a role in both the attachment of cells to basement membranes and, through its anionic properties, in the regulation of glomerular filtration (MYNDERSE et aI., 1983; RENNKE et a1. 1975). In addition to HSPG, proteoglycans with chondroitin and dermatan sulphate side chains have been found in basement membranes (BRENNAN et a1. 1984). 2.3.6 Type VII collagen

Type VII collagen is the major structural component of anchoring fibers (BENTZ et a1. 1983; SAKAI et a1. 1986). These horseshoe shaped structures originate from or insert into the lamina densa or else extend perpendicularly into the upper regions of the underlying connective tissue matrix (FARQUHAR and PALADE 1965; EADY 1988). Type VII collagen has been isolated from media of epidermoid cell cultures and could be identified as a molecule of at least 320 kD, consisting of a triple helical domain of 170 kD and with a 150 kD non-helical domain at the carboxy-terminus (LUNSTRUM et al. 1987).

In intact skin, type VII collagen occurs in anchoring fibers in association with amorphous dermal (anchoring) plaques, which also contain type IV collagen (BURGE SON eta1. 1985; KEENE et a1. 1987). Monoclonal antibodies have been raised against type VII collagen (SAKAI et a1. 1986; LEIGH et a1. 1987). These antibodies bind exclusively to (1) the dermal/epidermal junction, (2) sometimes the junctions of the epidermal adnexa and (3) the epithelial basement membranes in the esophagus and trachea. 2.3.7 Bullous pemphigoid antigens

Several investigators suggested that, in addition to the quantitatively dominant pro teins in the basement membranes listed in the previous paragraphs, many other minor basement membrane components might exist which have a more tissue or cell type specific distribution. Of these, the bullous pemphigoid antigen (BPA) has been one of the first to be identified. BPA is likely to play an important role in the attachment of the epidermis to the basement membranes - this is because in bullous pemphigoid, a blister ing skin disease, autoantibodies against BPA are deposited in the epidermal basement membranes. BPA could be identified on the basis of these antibodies; it has been characterized as a 220 kD molecule which may occur in vivo in at least a dimeric form (STANLEY et a1. 1986). The protein appears to be a component of hemidesmosomes (ANHALT et a1. 1987).


2.3.8 Basement membrane associated components

- Osteonectin, a Secreted Protein which is Acidic and Rich in Cysteine (also known by the acronym SPARC) is synthesized by cells which form large amounts of basement membrane material, e.g. F9 embryonal carcinoma cells and the EHS tumor (PAULS SON 1987). Osteonectin has been demonstrated by immunohistochemistry to be pre sent in Reichert's membrane and in the matrix of EHS tumor (DZIADEK et al. 1986) though not in all basement membranes. It is therefore not clear whether or not osteonectin occurs only in specialized basement membranes or, alternatively, is characterized by an ubiquitous distribution possibly masked by interactions with other basement membranes components. Osteonectin is certainly not an exclusive basement membrane protein because it occurs also in tissues rich in type I collagen such as bone and tendon. Osteonectin is a single chain polypeptide of about 40 kD with high calcium binding properties (PAULSSON 1987). - Type V collagen has been described as a basement membrane component (ROLL et al. 1980; MARTINEZ-HERNANDEZ and AMENTA 1983) although it is presently regarded as an interstitial rather than a basement membrane collagen. Type V collagen is not easily detected in the interstitium because of its intrafibrillar organization at the interior of type I and/or type III collagen fibers (BURGESON 1987; 1988; FITCH et a1. 1988). - Fibronectins are large cell adhesive glycoproteins (Mr of 500 kD) with a complex dimeric structure, occurring in plasma and in the extracellular matrix of connective tissue in the embryo and in healing wounds (McDONALD 1988). Many fibronectin isotypes exist due to differential splicing of a single pre-mRNA. Mammalian fibro blasts and also many other cell types possess specific fibronectin surface receptors which bind to an RGD sequence in the carboxyterminal part of the molecule (BucH and HOROWITZ 1987). Fibronectin is a basement membrane associated molecule rather than a true basement membrane component. Its occurrence is not confined to a single basement membrane layer. It can be found in a wide variety of tissues in the interstitial matrix (BROWNWELL et al. 1981; MARTINEZ-HERNANDEZ and AMENTA 1983). - Tenascin is a recently described large glycoprotein (Mr of 150-240 kD) which occurs in induced mesenchyme in the early morphogenetic stages of development of lung, kidney, gut, teeth, muscle, bone, skin and breast (CHIQUET-EHRISMANN et al. 1986). Its expression is variable and seems to be most intense at the time of maximal cellular interaction. In adult tissues it occurs in the peritendinous sheaths, perichondrium, nerve sheath and blood vessel wall. It has been suggested that tenascin functions as an antagonist to fibronectin, enhancing cellular mobility rather than cell adhesion (CHI QUET-EHRISMANN et al. 1988). In neoplasia, tenascin has been shown to be highly expressed in the stroma of malignant tumors but not in benign tumors (MACKIE et al. 1987). Although it is more an extracellular matrix component and only secondarily


associated with the basement membranes, tenascin is discussed here because it is apparently implicated in mesenchymal induction in neoplasia.

2.4 Basement membrane assembly

In connection with the isolation and characterization of the various basement mem brane components, various models have been developed for the supramolecular struc ture of the basement membrane. There appears to be a consensus concerning the basic structure of the basement membrane as a dense network of fine cords (INOUE et al. 1988). The cords consist of an intensely cross-linked network of type IV collagen, which provides the mechanical stability. It is conceivable that the three dimensional alignment of triple helical collagen type IV molecules results in a polygonal structure. Various models have been proposed for the three dimensional assembly of basement membranes. The oldest model depicts the basement membranes as a layered structure with alternating strata of collagen and non-collagenous proteins interconnected at specific sites so as to create a three dimensional structure (SCHWARTZ and VEIS 1980; SCHWARTZ et al. 1980). This model is supported by the tendency of type IV collagen, laminin and heparan sulphate proteoglycan to form homopolymers. Further evidence in favor of this model has been provided by immunoelectron microscopic studies, which demonstrated that anionic sites (representing heparan sulphate proteglycans) are con centrated on the basement membrane surface (KANWAR and FARQUHAR 1979 a,b)) and that distinct stratifications of reactivity within the basement membrane co-exist with antibodies raised against laminin and heparan sulphate proteoglycan. The matrisome model, proposed by MARTIN et al. (1984) is based upon the tendency of the different components to form stable complexes exhibiting characteristic stoichiometry. In this model the secreting cell produces complexes of laminin, entactin/ nidogen and heparan sulphate proteoglycan, which spontaneously polymerize due to the high affinity of these components for each other. After deposition, the matrix is rearranged to include molecules of type IV collagen in the network structure. The codistribution of laminin, entactin/nidogen and heparan sulphate proteoglycan sup ports the matrisome model. One of the later models, proposed by FURTHMAYER et al. (1985), postulates the tendency of the different components to form hetero- or homopolymers and has been named the polymorphic polymerization model. An attractive aspect of this model is that it leaves room for differences in basement membrane structure at different anatomi cal sites, those differences being referred to variations in the synthesis and deposition of the individual components. Basement membrane heterogeneity - in terms of quantita tive differences in the concentration of a particular component at different sites as well as differences in the supramolecular structure - has been repeatedly reported (BOSMAN et al. 1989).

Basement membranes in neoplasia· 15

The most recent publications indicate that the actual structure of the basement mem brane may exhibit aspects of all three models. YURCHENKO and RUBEN (1987) recently studied amnionic basement membranes en face in the native state and after salt extrac tion. They conclude that the basement membrane consists of a type IV collagen net work, which is a layered polygonal of laterally associated branching monomolecular filaments with globular structures and which represents the dimeric NC1-terminal domains. The network appears to be highly similar to that spontaneously formed by type IV collagen in vitro through carboxyl-terminal dimerization, lateral association with superhelix formation and aminoterminal 7$ formation (YURCHENKO and $CHNITI NY 1990). It can therefore be argued that cells form a basement membrane scaffold by secreting type IV collagen into a confined compartment where the network then forms spontaneously. It is not unlikely that cell- or tissue type specific variations in the mesh structure are introduced by variations in the local conditions (pH, salts, protein concen tration, modulating effects of other extracellular matrix molecules) brought about by the producing cells. The laminin polymer is either sequentially or simultaneously formed through interaction between the end of the short arm, the two polymers being attached through entactin/nidogen bridges and directing low-affinity laminin-collagen interactions.

3 Interaction of neoplastic cells with basement membranes The classical view of the role of basement membranes in neoplasia regarded these 6tructures as passive mechanical barriers against the invasive neoplastic cells. More recent data have indicated, however, that basement membranes are not only degraded by invading tumor cells but may also be deposited in a growing neoplasm, either by the tumor cells or by the reactive tumor stroma. It has furthermore been shown that specific interactions occur between neoplastic cells and basement membrane components and that these interactions might play an important role in the metastatic process. Finally, extracellular matrix components provide specific stimuli for tumor cell differentiation. In this Chapter, these aspects of the interaction between neoplastic cells and basement membranes will be reviewed.

3.1 Basement membrane degradation in invasive growth Tumor invasion can be defined as the extension of neoplastic cells beyond the natural borders of the tissue or cell type from which they derive. As a result of this active process of infiltrative growth the neoplastic cells occur in tissue compartments where they do not belong. In an invasive carcinoma, for example, the malignant cells penetrate through the epithelial basement membrane and proliferate into the surrounding mesen


chymal stroma. For the formation of metastases, the tumor cells have to penetrate through a lymph or blood vessel wall and, after formation of a tumor cell embolus in the circulation, the tumor cells again have to pass through the vascular endothelial basement membrane before a metastasis is formed (Fig. 7). This sequence of events illustrates that in the pathogenesis of neoplasia basement membranes playa rather central role.

- --

Fig. 7. Schematic representation of the various steps involved in the development of a tumor metastasis. a: penetration of the epithelial basement membrane, b: migration into tumor stroma, c: penetration of vascular basement membrane, d: migration in the circulation, e: lodging at a metastatic site and penetration of the local vascular basement membrane, f: outgrowth of the metastasis.


There has been a long-standing interest in the role of the basement membrane in neoplastic growth. The earliest studies were performed using the reaction which selec tively stains the basement membrane because of its high glycoprotein content. The PAS reaction, however, does not only detect basement membrane glycoproteins, thus reticu lin silver staining has also been used to stain basement membranes. Electron microscopy too has been used extensively to study basement membranes in neoplasia. One of the first reports (ZELICKSON 1962) described the presence of intact and continuous base ment membranes around basal cell carcinomas of the skin. Subsequent studies have contested this finding (KOBAYASHI 1970; MCNuTT 1972) and in fact subsequent ultra structural studies have shown that in most carcinoms the neoplastic cells either lack a basement membrane or display a highly discontinuous basement membrane (GOULD et al. 1975; GOULD and BATTIFORA 1976). Well differentiated carcinomas generally tend to retain a recognizable basement membrane structure, whereas in undifferentiated car cinomas the basement membranes appear to be scanty or absent. In carcinomas in situ or in «borderline» malignant lesions interruptions or gaps in the basement membranes tend to occur (GOULD and BATTIFORA 1976). In recent years immunohistochemistry has greatly expanded the avenues for studying basement membranes in cancer. Here the availability of antibodies specific to a variety of basement membrane components has been the key factor. This approach circumvents one of the important limitations of electron microscopy in the analysis of large tissue samples and therefore of general tissue architecture. The ultrastructural findings have mostly been corroborated by immunohistochemical studies. With immunohistochemis try almost all benign lesions show an intact basement membrane, whereas most malig nant epithelial lesions show basement membrane discontinuities, varying down to total absence of the basement membranes in some tumors. Again, in borderline malignant lesions or in situ carcinomas discrete gaps or discontinuities in the basement membranes can be observed (BOSMAN et al. 1985). Exactly how basement membranes are penetrated by invasive tumor cells has been the subject of intensive study in recent years and useful model systems for invasive growth have been developed (MAREEL et al. 1987). Originally it was thought that the presssure of expansively growing cells would be enough to push the tumor cells through the membrane (EAVES 1973). It has become clear, however, that a complex process of proteolyic digestion is responsible for the basement membrane breakdown (YEE and SHIU 1986; TRYGGVASON 1989). High activity of proteolytic enzymes has been demons trated at the site of tumor invasion (BARSKY et al. 1983 a, b); these enzymes are regarded as responsible for the dissolution of the basement membrane because proteinase in hibitors can block the invasive process (THORGEIRSSON et al. 1982; GIRALDI et al. 1984). An important question is whether tumor cells themselves produce these enzymes or whether, alternatively, resident or reactive cells (stromal fibroblasts, macrophages) are responsible for their production. Recent studies have convincingly shown that tumor cells themselves produce basement membrane degrading proteinases (LIOTTA et al.


1979, 1980; SALO et al. 1982; STARKEY et al. 1984; MONTEAGUDO et al. 1990). This does not imply, however, that basement membrane degradation occurs exclusively through tumor cells. Proteinase degradation of basement membranes has appeared to be a rather specific process. It is evident that degradation of type IV collagen, which constitutes the base ment membrane scaffold, is of crucial importance (NAKAJIMA et al. 1987). Type IV collagen is highly resistant to degradation by interstitial (types I and III) collagenases but reasonably susceptible to degradation by pepsin (TRYGGVASON and KIVIRIKKO 1978), cathepsins Band G (DAVIES et al. 1978), leucocyte elastase (MAINARDI et al. 1980), trypsin (SCHUPPAN et al. 1984), and chymotrypsin (TRYGGVASON et al. 1984). LIOTTA and coworkers were the first to detect type IV collagen specific proteases (LIOTTA et al. 1979; SALO et al. 1982). Subsequently others (TSUDA et al. 1988) have also reported the existence of type IV collagen specific metalloproteases. The type IV col lagenases appear to be produced as inactive precursors that are then activated by cleav age via the plasminogen activator - plasmin system (SALO et al. 1982; STETLER-STEVEN SON et al. 1989). How the production of type IV collagenase is regulated is largely unknown. Production of the enzyme can be induced by exposing fibroblasts to tumor promoting phorbol esters (SALO et al. 1985). As has been indicated, it is likely that, in addition to type IV collagenase, the plas minogen activator - plasmin system also plays a role in basement membrane dissolution by neoplastic cells (REICH et al. 1988). Plasminogen activators of both the urokinase type and the tissue type have been found in most transformed cells and malignant tissue (DANO et al. 1985). Plasmin cannot degrade type IV collagen but may be involved in the activation of type IV collagenase (SALO et al. 1982). Furthermore, plasmin can degrade both laminin and fibronectin (BALIAN et al. 1979; LIOTTA et al. 1981). Among the cathepsins (a family of acid lysosomal sulphhydryl proteinases) cathepsin B has been found in elevated amounts in neoplastic cells (POOLE et al. 1980; SLOANE and HONN 1984). Cathepsin may degrade type IV collagen and may also be involved in the lysis of laminin and proteglycans. Another category of enzymes presumably involved in base ment membrane dissolution is that of the glycosidases. These enzymes may be specifi cally involved in the breakdown of the glycosaminoglycan side-chains of the basement membrane proteoglycans (KRAMER et al. 1982). Heparanases might also be involved (NAKAJIMA et al. 1988).

3.2 Basement membrane deposition by neoplastic cells Although initially basement membranes in neoplasia were primarily studied with regard to their dissolution in invasive growth, it has become increasingly clear that the deposition of an extracellular matrix and, more specifically, of basement membranes is a quite important determinant of the behavior of the neoplasm (BARSKY et al. 1988). It has


been known for a long time that some tumors are characterized by an abundant stroma. In histopathology this phenomenon has become known as the «desmoplastic reaction». This reaction consists of a remarkable proliferation of stromal cells (e.g. fibroblasts, myofibroblasts and vascular cells) which deposit an abundant extracellular matrix. Typ ical examples of such a neoplasm are «scirrhous» carcinoma of the breast and desmo plastic adenocarcinoma of the lung. Both ultrastructural studies (GOULD and BATTIFORA 1976; HAVENITH et al. 1988, 1989) and immunohistochemical studies (BOSMAN et al. 1985) have shown that base ment membrane material can be deposited around nests of neoplastic epithelial cells, often in irregular patches but occasionally as a regular basement membrane-like struc ture. A characteristic example of neoplastic basement membrane deposition is found in the adenoid cystic carcinoma, in which a dense multilayered basement membrane is formed around tumor cell nests (Fig. 8). Another characteristic example is malignant melanoma. Even around overtly invasive fields of melanoma cells a basement membrane

8. Basement membrane deposition in adenoid cystic carcinoma of salivary gland. Note multi amelar immunoreactivity around epithelial cells nests (curved arrows) and intracytoplasmic im nunoreactivity (open arrows). - Type IV collagen immunoperoxidase (x 250). ~ig.


Fig. 9. Basement membrane deposition in metastatic melanoma in a lymph node. - Type IV collagen specific immunoperoxidase staining (A = x 100, B = x 250).

can usually be detected (HAVENITH et al. 1989). Also in metastatic sites (e.g. in lymph node metastasis) deposition of basement membranes can be observed (Fig. 9). This phenomenon does not only occur in epithelial neoplasms, but also in mesenchymal neoplasms arising from basement membrane producing cells (e.g. vascular, muscle and fat cell tumors). An important question is how these basement membranes are deposited. It was long assumed that epithelial basement membranes are exclusively produced by the adjacent epithelial cells. It has gradually become clear, however, that the deposition of a base ment membrane arises through interaction between an epithelial cell and its surround ing stroma. Several studies have used immunohistochemistry to show this conclusively on xenografts of human tumors employing species (mouse or human) specific anti bodies araised against laminin. DAMJANOV et al. (1985) found that in the tumor stroma the vascular basement membranes contained mouse laminin, whereas the epithelial basement membranes were of human but occasionally also of mouse or mixed origin.

Basement membranes in neoplasia' 21

;ig.l0. Type IV collagen immunoreactivity in human tumor cell xenografts in nude mice as evealed by species specific anti-type IV collagen antibodies. A-C epidermoid carcinoma cell line KB); D-F colon carcinoma cell line (5583). Immunohistochemistry with species cross reactive A, D) human specific (B, E) and mouse specific (C, F) anti type IV collagen antibodies. In all :enografts basement membranes are deposited but some with only mouse (stromal) type IV ollagen (E, F) but in others with mouse (stromal) and human (epithelial) type IV collagen (B, C). . Immunoperoxidase (x 250).


Fig. 11. Detection by in situ hybridization of type IV collagen mRNA in nude mouse xenografts of colon carcinoma (5583) cells (A, B) or human epidermoid carcinoma (KG) cells (C, D). The 5583 xenografts show only stromal labeling whereas the KB xenografts show epithelial cell label ing also. - 35S Autoradiography (x 250).


Similar experiments were performed by CLEUTJENS et a1. (1990). They used immunohis tochemistry to study human tumor xenografts, employing species (mouse or human) specific antibodies raised against type IV collagen. Xenografts induced by inoculating tumor cells that showed in vitro production of type IV collagen were found to contain mouse and human type IV collagen epitopes (Fig. 10). When tumor cells lacking the :apacity to produce type IV collagen were inoculated, the basement membranes turned 3ut to be exclusively of mouse origin. These results were confirmed by in situ hybridi lation, using cDNA probes for type IV collagen mRNA (Fig. 11). These findings indicate that the basement membrane is a joint product of epithelial and stromal cells. It is most likely that the tumor cells secrete regulating proteins which induce the produc tion of an extracellular matrix in the tumor stroma. Conversely, the tumor stroma may Jlay a role in tumor cell differentiation. Which mechanisms regulate these processes is .argely unknown. From a practical point of view, the production of basement membrane material by :umor cells is of some importance in diagnostic histopathology. Several studies have Jeen published on basement membrane patterns in soft tissue tumors (MIETTINEN et a1. 1983; D'ARDENNE et a1. 1984; OGAWA et a1. 1986). In principle, these tumors can be :livided into those that do not (fibrosarcoma, malignant fibrous histiocytoma) and those :hat do (angiosarcoma, myosarcoma, liposarcoma, etc.) produce basement membrane naterial (BOSMAN et a1. 1990). In carcinomas generally, the extent of basement mem )rane deposition seems to reflect the degree of differentiation of the tumor. In well :lifferentiated carcinomas extensive basement membrane deposition can frequently be 10ted, whereas in poorly or undifferentiated carcinomas basement membranes are usu Illy lacking. Several investigators have established a correlation between prognosis and he extent of basement membrane deposition. In colon carcinoma and in squamous cell :arcinoma of the lung this has been found to be of significant prognostic importance, umors with extensive basement membrane production being characterized by longer nedian survival (HAVENITH et a1. 1988; TEN VELDE et a1. 1991). Similar findings were 'eported for bladder cancer (DAHER et a1. 1987; SCHAPERS et a1. 1990).

U Cellular attachment to basement membranes

Normal epithelial cells develop extensive adhesive contacts with their environment. rhey mutually form morphologically distinct junctions, such as desmosomes and tight unctions. They appear to adhere firmly to the adjacent basement membrane. The ntercellular adhesions are not relevant in this context, although the loss of intercellular :ontacts appears to be a crucial element in the development of the neoplastic phenotype .nd one of the factors responsible for its propensity for invasive growth. Cells mostly adhere to the extracellular matrix with the aid of glycoprotein attach nent factors. These molecules form connections between receptors on the cell surface


and specific binding sites on the structural components of the basement membranes (especially type IV collagen). It is conceivable that the cells themselves produce attach ment factors. An alternative possibility is that the cells attach to factors present in the extracellular matrix. A combination of these two possibilities is also possible or even most likely. Specific attachment factors have been identified. Fibronectin and laminin have been long known. It has been recently established that a family of more or less closely related attachment proteins exists. In view of their integrating function these proteins have been designated integrins (RUOSLAHTI and PIERSCHBACHER 1987). Fibronectin is, of the above mentioned proteins, the one best characterized compo nent (HYNES and YAMADA 1982). The fibronectin gene has been cloned and close scrutiny of the functional characteristics of the polypeptide chain has shown that the molecule contains a number of discrete binding sites for cell surface receptors and for extracellular matrix proteins such as laminin and heparan sulphate proteoglycan. The function of fibronectin is not restricted to adhesion, however. Fibronectin organizes various components of the extracellular matrix and can transmit signals to the cell. A significant problem in attempts to elucidate the role of fibronectin in vivo is the fact that it is a very ubiquitous molecule. Highly abundant in the serum, it is also produced in high quantities by stromal cells and released into the extracellular matrix. But the exact function of fibronectin in the interaction between neoplastic cells and basement memb ranes is not clear. Due to its fairly restricted occurrence, the role of laminin could be more effectively studied. As has been noted earlier, type IV collagen binding sites occur in the globular domains of both the short and the long arms (RAo et al. 1982; LAURIE et al. 1986). Furthermore, severallaminin specific binding sites have been identified on the surface of normal and neoplastic cells (TERRANOVA et al. 1980, 1983). A laminin receptor was first identified on the surface of MCF-7 breast carcinoma cells (TERRANOVA et al. 1983). Subsequent studies have shown that this receptor is a protein with a molecular weight of about 67 kD; this occurs not only on MCF-7 cells but also on mouse melanoma and fibrosarcoma cells (MALINOFF and WICHA 1983; VARANI et al. 1983). Later studies have shown that in fact several specific laminin binding cell-surface proteins exist (RAo et al. 1982, 1983; COUCHMAN et al. 1983; LIOTTA et al. 1984). It is not known whether or not internalization of these proteins occurs subsequent to their binding with laminin. The term receptor for these proteins may therefore be inappropriate. It is clear, however, that laminin binding does not merely involve passive attachment of cells. It may also induce cellular differentiation (WICHA et al. 1982; KLEINMAN et al. 1985; KLEIN et al. 1988). It has been postulated that, in the early phases of the development of metastasis, laminin may play a role in the attachment of circulating tumor cells to the vascular basement membrane at the metastatic site. According to this hypothesis, cells without a laminin receptor would metastasize less efficiently than cells with a laminin receptor. Differential expression of laminin receptor by high- and low-metastatic tumor cells was


confirmed in a study conducted by VARANI et a1. (1983). However, it has also been reported that breast cancer with laminin receptor expression behaves less aggressively than breast cancer without laminin receptor expression (MARQUES et a1. 1990). It might be anticipated that the treatment of tumor cells with an antibody raised against the laminin receptor, or with a peptide sequence which reacts with the laminin receptor, might decrease the metastatic capacity of neoplastic cells. Along this line, IWAMOTO et a1. (1987) demonstrated that YIGSR, a synthetic laminin pentapeptide, has an inhibiting effect on the development of experimental melanoma metastasis. During lodging of neoplastic cells in the circulation, tumor cell - laminin interaction cannot be envisaged as a primary event. Circulating cells never encounter laminin because the surface of most of the vascular system is covered with a closed endothelial layer. KRAMER et a1. (1980) and KRAMER and NICHOLSON (1981) have shown that after the attachment of tumor cells to the endothelial surface the endothelial cells retract, thus exposing the underlying basement membrane. In this phase of the metastatic process the platelet membrane might be implicated (MENTER et a1. 1987). Laminin binding may then playa role in the migration of the metastasizing cell through the vascular wall.

t4 Modulation of cellular growth and development by basement membrane components As has been mentioned previously, basement membranes do not only serve as passive tttachment sites for epithelial cells but may also induce cellular differentiation and :unctional activity. . It has been reported that extracellular matrix, including laminin and type IV collagen, nduces differentiation in rat small intestinal epithelial cells (CARROLL et a1. 1988) and in nammary epithelial cells (WICHA et a1. 1982). In a recent report HAHN et a1. (1990) lescribed the induction of differentiation in rat intestinal epithelial cells in vitro by "econstitued EHS sarcoma derived basement membrane material as well as by purified aminin and type IV collagen. Similar findings were reported by WALLING et a1. (1991) :or MAC 15 murine colonic adenocarcinoma cells. These findings indicate that tumor :ells do not behave autonomously but are subject to phenotypic modulation by their ~nvironment (BOYD et a1. 1988). Epithelial cells have further been found to produce >asement membrane proteins but only when in contact with extracellular matrix pro eins. This has been reported for corneal epithelium (DODSON and HAY 1974) thyroid :pithelial cells (GARBI and WOLLMAN 1982; GARBI et a1. 1988), fibroblasts (COUCHMAN :t a1. 1983) and neural cells (TOMASELLI et a1. 1987). The basement membrane evidently nodulates the functional differentiation of many different cells and is also involved in he regulation of the expression of genes coding for basement membrane components. rumor cells may also respond to these regulatory mechanisms. This suggests that not mly the intrinsic characteristics of tumor cells but also their response to modulating actors from the host determines the biological behavior of the tumor.



Basement membrane histochemistry: Methodological aspects 1.1 Conventional histochemistry and electron microscopy The study of basement membranes is not entirely new. Initially basement membranes ;vere stained using the periodic acid Schiff (PAS) reaction. Using this technique the )asement membrane, which contains a high concentration of glycoproteins such as aminin and heparan sulphate proteoglycan, stains a regular line demarcating the adja :ent cell (Fig. 12 A). This approach is still fairly popular in the analysis of basement nembrane characteristics in glomerular diseases of the kidney. The PAS technique, lOwever, stains a wide variety of glycoproteins (in addition to glycogen) and therefore acks specificity for the basement membrane. Another approach is the use of silver mpregnation techniques, including the silver-methenamine, the Gordon and Sweet and he Jones procedures (Fig. 12 B). These techniques impregnate collagens, especially the "ine fibrillar type I collagen fibers but also the basement membrane. Silver impregnation s also a frequently used technique for staining basement membranes in renal biopsies mt may also be used to delineate basement membranes in neoplasia (PUCHTLER and IWEAT 1964). As in the case of the PAS reaction, this technique lacks specificity, and herefore does not allow detailed analysis of basement membrane morphology or com ,osition. As has been previously mentioned, more specific information regarding basement nembrane characteristic in neoplasia has been obtained by ultrastructural studies. Al hough electron microscopy has not allowed chemical analysis of the basal lamina, its norphology has been adequately analyzed and detailed studies on basement membrane llterations in neoplasia were performed by GOULD and BATTIFORA (1976). These au hors described the absence of basement membranes in areas of invasive cancer, but also ·eported the deposition of morphologically irregular basement membrane-like struc ures by neoplastic cells. A major limitation of electron microscopy, however, is the :mall size of the specimens that can be analyzed. As the basement membrane configura ion can differ significantly from one area to another in the same tumor, many tissue ,locks have to be analyzed in order to obtain a reasonably reliable overall impression of he status of the basement membrane in a particular neoplasm. This problem is largely llleviated by the immunohistochemical approach, using antibodies specific for base nent membrane components. In fact, a combination of ultrastructural analysis and mmunohistochemistry appears to be the optimal approach (APAJA-SARKKINNEN et al. 986; HAVENITH et al. 1990).

;ig. 12. Basement membranes in the kidney as revealed by PAS staining (A) Jones' silver impreg lation (B), and type IV collagen immunohistochemistry (C). - (x 250).


4.2 Immunohistochemistry For reliable immunohistochemistry, optimal conditions for tissue processing have to be established and antibodies specific for relevant basement membrane components have to be available. 4.2.1 Tissue processing procedures

For basement membrane immunohistochemistry a wide range of tissue processing procedures have been applied. The least demanding approach is the use of unfixed fro~en sections which after drying may be used after brief fixation in cold (-20°C) acetone or ethanol. Basement membrane components are fairly insoluble and relatively stable and can therefore be very easily stained in cryostat sections. For routine purposes, however, the use of paraffin sections is preferable. Brief fixa tion in 4% buffered formaldehyde (HAVENITH et al. 1987), is compatible with reliable immunohistochemistry. Any cross-linking fixative, however, rapidly diminishes the immunoreactivity of basement membrane antigens, especially of type IV collagen and laminin. Prolonged fixation should therefore be avoided. For routine purposes, fixation in phosphate buffered 4% formaldehyde for 3-6 h is a reliable approach. For im munoelectron microscopy, periodate-Iysine-paraformaldehyde (McLEAN and NAKANE 1974) or glutaraldehyde-paraformaldehyde is a useful alternative. After aldehyde fixation, the immunoreactivity of laminin can be at least partly re stored by digestion of the sections after rehydration in 0.4% pepsin in 0.02 mHCl (EKBLOM et al. 1982). The optimal duration of the pepsin treatment as mentioned in the literature is somewhat variable (between 2 and 9 h at 37°C or at room temperature). This variation may be due to differences in the fixation conditions and in the specific activity of the enzyme batch. As a consequence, the optimal conditions for pepsin treatment have to be established for each laboratory. Under appropriate conditions, and assuming that the tissue was not overfixed, the majority of paraffin embedded tissues can be reliably stained for laminin. In some specimens restoration of laminin im munoreactivity cannot be attained. A somewhat similar situation exists for type IV collagen. Here too the immunoreac tivity of this basement membrane component can be restored by pepsin treatment, using basically the same approach as for laminin. Also for type IV collagen the optimal conditions of pepsin treatment will have to be established for each laboratory. We have established the usefulness of tissue pretreatment in 4M guanidine HCI before treatment with pepsin for restoration of type IV collagen immunoreactivity (HAVENITH et al. 1989). Type IV collagen immunoreactivity can be restored in the large majority of paraffin embedded tissue specimens using this techniqe. This approach does not work for laminin, however. In view of this fact, we prefer to use type IV collagen immunohis tochemistry for the routine assessment of basement membrane characteristics in his topathological analysis.


For immunoelectronmicroscopical purposes different requirements have to be met. [n order to preserve tissue morphology fixation has to be adequate, pepsin treatment cannot be used, yet sufficient immunoreactivity of basement membrane components has to be retained. Early immunoelectronmicroscopical studies mostly employed PLP fixation as part of indirect peroxidase labelled antibody techniques and also made use of pre-embedding methods (MARTINEZ-HERNANDEZ et al. 1982; LAURIE et al. 1984). Sub sequent studies have shown that mild fixation can be combined with low temperature resin embedding to allow postembedding immunoelectron microscopy, i.e. using the Lmmunogold technique (HAVENITH et al. 1990). The latter approach has the advantage of higher resolution: it is easier to establish the precise localization of a 5 nm gold par ticle than an irregularly outlined electrondense product of the osmicated diaminoben lidine reaction. The advantage of the pre-embedding technique, using the peroxidase labelled antibody approach is that larger tissue specimens can be examined by light microscopy before selected areas are reembedded for ultrathin sectioning. tl.2 Antibody specificity

A wide variety of antibodies have been used for specific immunostaining of the basement membrane, including antibodies directed against type IV collagen, laminin, ~eparan sulphate proteoglycan and entactin/nidogen. For general purposes, however, type IV collagen and laminin have been the prefered antigens and therefore we will limit :he discussion to antibodies raised against these proteins. The first anti-Iaminin antibodies described (FOIDART et al. 1980) were polyclonal ;rabbit) and raised against laminin isolated from EHS sarcoma. These antibodies have Jeen extensively used in a multitude of reports. Subsequently, several monoclonal mtibodies raised against mouse laminin (WAN et al. 1984) and against human laminin :ENGVALL et al. 1986) have been reported. Many of the monoclonal anti-Iaminin anti Jodies cannot detect laminin in formalin fixed tissue specimens. Furthermore, species ;pecificity or specificity for molecular subtypes of laminin, with a restricted tissue :listribution (WAN et al. 1984; WEWER et al. 1987) has been described for monoclonal mti-Iaminin antibodies. For routine purposes, therefore, polyclonal anti-laminin anti Jodies are preferable. A fairly extensive collection of poly- and monoclonal antibodies specific for type IV :ollagen is now available. Most polyclonal antisera are not species specific, although ~abbit antisera against mouse type IV collagen have been described recently (CLEUTJENS ~t al. 1990), which after absorption with human type IV collagen reacted only with rat md mouse basement membranes. For routine basement membrane immunohis :ochemistry these polyclonal antibodies are entirely satisfactory. The first monoclonal antibodies to human type IV collagen that were species cross 'eactive were produced by SUNDARRAJ and WILSON (1982). SAKAI et al. (1982) described I human specific anti-type IV collagen antibody. Subsequently a variety of antibodies


has been described, often with a species-restricted reactivity and sometimes specific to the species of origin of the used immunogenic material (FOELLMER et a1. 1983; ODER MATT et a1. 1984; SCHEINMAN and TSAI, 1984; HAVENITH et a1. 1989). Some monoclonal antibodies react with native epitopes which cannot be detected in processed tissues (CLEUTJENS et a1. 1990). Many antibodies, however, detect epitopes which can be detected in processed tissues, usually after pepsin and/or 4M guanidine HCI pretreat ment (HAVENITH et a1. 1989). 4.3.2 Choice of label For light microscopical studies, the usual fluorescent labels (fluoresceine and rhodamine) have been used with the same success as peroxidase. For aldehyde fixed tissues the peroxydase labelled approach is preferable because of the occurrence of autofluorescence, which however does not appear to be an overwhelming problem due to readily recognizable linear configuration of the basement membrane. With both types of label double labeling can be performed. Peroxidase labels have the traditional advantage of yielding permanent sections.

Fig. 13. Immunoelectronmicroscopicallocalization of laminin in the tubular basement membrane (bm) in the kidney. Note intense immunoreactivity at the side of the lamina lucida adjacent to the cell surface and extending into plasmamembrane invaginations into the cell-(scale bar = 0.1 I-lm).


For electron microscopy, peroxidase labelling in a pre-embedding approach has been Llsed extensively (MARTINEZ-HERNANDEZ 1982, 1984 a, b). An advantage of this ap Jroach in its simplicity and reliability: with appropriately fixed tissues immunoreactivi :y is not a problem and acceptable ultrastructure can be obtained. However, antibody Mfusion in the tissue might be limited by basement membranes and this possibility has .ed to controversy over the reality of the observed higher intensity of laminin im nunoreactivity in the lamina rara of the glomerular basement membrane (ABRAHAMSON ~t al. 1986; LAURIE et al. 1984) (Fig. 13). More recently, postembedding techniques Llsing low temperature (Lowicryl) resin embedding in combination with colloidal gold abelling have been employed. Using this approach, tpye IV collagen and laminin have )een repeatedly localized in basement membranes using immunoelectronmicroscopy :MARTIN and TIMPL 1987).

; Basement membrane immunohistochemistry as a tool in in the histopathological diagnosis of cancer ;.1 General principles As has been outlined in chapter 3, an important characteristic of malignant neoplasia s the occurrence of invasive growth. Non invasive neoplasms are benign or, at most, )remalignant. Invasive neoplasms are almost always malignant. Recognition of these ~rowth patterns constitutes the most important application of basement membrane mmunohistochemistry in diagnostic pathology. There are, however, two other applica :ions. Firstly, the tendency of neoplasms to deposit a basement membrane seems to be lssociated with differentiation of the neoplasm: e.g. in carcinomas a correlation appears :0 exist between degree of differentiation and tendency to deposit a basement mem )rane. Secondly, in soft tissue sarcomas basement membrane deposition permits dif :erentiation of two broad categories of neoplasm. •.1.1 The basement membrane and invasive growth

As has been pointed out above, it is important in diagnostic histopathology to distin ;uish between neoplasms that do and those that do not display invasive growth charac eristics. Frequently this appears not to be a problem. In certain organs, however, some leoplasms develop gradually from a benign to a premalignant to finally a malignant ;tate. Squamous cell carcinoma of the uterine cervix is a typical example, developing :rom dysplasia via carcinoma in situ to finally invasive carcinoma. With these lesions it :an be rather difficult to establish for a particular lesion whether or not invasion has )ccurred. In this situation basement membrane immunohistochemistry can be useful. rhis approach has been advocated in cervical, laryngeal, and epidermal squamous cell


carcinoma and in adenocarcinomas of the digestive tract. It is still too early to determine whether or not basement membrane immunohistochemistry leads to more accurate diagnosis. Follow-up studies comparing immunohistochemical data with clinical infor mation should establish whether or not interruptions in the basement membrane in lesions without histological signs of invasion permit identification of an early stage of invasive growth. It is already clear, however, that basement membrane interruptions are not always indicative of invasion in a neoplasm. VISSER et al. (1986) showed in laryngeal neoplasms that an inflammatory reaction in a non invasive lesion can result in basement membrane interruptions. Conversely, the presence of a basement membrane does not exclude invasive growth, as neoplastic cells may effectively deposit this material. Strik ing examples are malignant melanoma, thyroid carcinoma and adrenal carcinoma (Fig. 14). This ambiguity may be the reason why basement membrane immunohistochemistry has not been effectively used to study borderline lesions. These lesions, which have been reported in several organs but most characteristically in the ovary, in fact constitute a no-man's land between benign and malignant categories, leaving pathologists without

Fig. 14. Basement membrane deposition in malignant melanoma (A) and follicular carcinoma of the thyroid (B). Immunoperoxidase staining for type IV collagen. - (A = x 240, B = x 100).


;ufficient histological clues to predict the behavior of the lesion. Our initial results with )Varian lesions (unpublished observations) suggest that ovarian borderline lesions might )e subdivided into a group that shows a basement membrane pattern comparable to that )f benign cystic adenomas (regular, continuous) and into a group with a focal pattern -esembling that of adenocarcinomas (irregular, discontinuous). More studies of patient naterial, including follow-up data, will be required if the potential usefulness of this lpproach is to be demonstrated. i.1.2

Basement membranes and differentiation in carcinomas

As has been pointed out in chapter 3, the deposition of basement membrane material s not orchestrated by the carcinoma cell alone but results from interaction between :arcinoma cells and stromal cells (e.g. myofibroblasts). As a consequence, there appears o be no unequivocal relationship between tumor cell differentiation and basement nembrane deposition, despite a general tendency by well differentiated lesions to de )osit more basement membrane material than poorly differentiated lesions. In the )reast, for example, tubular carcinomas fall into the category of well differentiated :arcinomas but often totally lack basement membranes. By contrast, in colon carcinoma veIl differentiated tumors often deposit more basement membrane material than poorly lifferentiated tumors, though in individual cases striking exceptions may be encoun ered. The tendency of a neoplasm to deposit basement membrane material has been dentified as a prognostically favorable sign in cancer of the colon (FORSTER et al. 1984, 986; HAVENITH et al. 1988), lung (TEN VELDE et al. 1991) and bladder (CONN et al. ,987; DAHER etal. 1987, SCHAPERS et al. 1990). Some carcinomas show a highly characteristic pattern of basement membrane deposi ion. Adenoid cystic carcinomas, for example, show concentric lamellar deposition of lasement membrane material, coinciding with the histologically identifiable :osinophilic and strongly PAS positive band surrounding tumor cell nests. In squamous ell carcinoma of the lung the tumor periphery usually displays an intact alveolar lattern, which indicates that the tumor is growing intraalveolarly, leaving the preexist ng tissue architecture largely intact (DINGEMANS and MoOI] 1987; HAVENITH et al. 989). ,.1.3

Basement membranes and classification of soft tissue sarcomas

Many mesenchymal cells, including muscle, fat, Schwann cells and endothelial cells re actively involved in the deposition of a basement membrane. It is therefore not urprising that tumors derived from these cell types can be distinguished on the basis of lasement membrane production. Mesothelioma and synovial sarcoma form an important but separate category. These umors have histological characteristics of sarcoma as well as of carcinoma. In the


sarcomatous areas, linear basement membrane deposits can be found between the spin dle shaped tumor cells. In the epithelial areas, the tubular or slit like configurations are usually surrounded by a basement membrane. Basement membrane immunohis tochemistry highlights these architectural characteristics and can therefore be diagnosti cally helpful. Fibrosarcomas and malignant fibrous histiocytomas (MFH) constitute categories of neoplasms which do not deposit basement membrane material, although in the case of MFH some controversy exists (SOINI 1989). This is due to the lack of a distinct set of criteria unambiguously identifying MFH. In diagnostic practice MFH appears to be more of a «leftover» category of tumors which, ultrastructurally or immunohistochemi cally, exhibit a wide spectrum of differentiation characteristics. Other soft tissue sarcomas (liposarcoma, hemangiosarcoma, malignant Schwannoma, leiomyosarcoma, rhabdomyosarcoma) appear to deposit basement membrane material. As in carcinomas, however, the extent appears to depend somewhat on the degree of differentiation. In poorly differentiated neoplasms identification of basement mem brane material is often impossible. Given this situation, basement membrane Im munohistochemistry may not be of significant help in classifying the neoplasm.

Fig. 15. Basement membranes surrounding adipocytes in the subcutis. - Type IV collagen im munohistochemistry (x 100).


5.2 Skin

In the normal human skin, immunohistochemical localization of type IV collagen and laminin discloses a regular and continuous basement membrane location both at the dermo-epidermal interface and around epidermal adnexa, including hair follicles, sebaceous glands and sweat glands (GUSTERSON et al. 1984, 1986; STENBACK and WASENIUS 1985). In the dermis smooth muscle fibers, in arrector pili muscle and in the media of vessel walls, Schwann cells surrounding nerves and the endothelial lining of blood vessels are

'~'! Fig. 16. Type IV and type VII collagen distribution in laryngeal mucosa. Type VII collagen (A), the main component of anchoring fibrils occurs only in the basement membrane zone of the surface squamous epithelium and around a duct (open arrow). Type IV collagen occurs in all basement membranes (B). - Immunoperoxidase (x 60).


stained (Fig. 1). In the subcutis fat cells are surrounded by a basement membrane (Fig. 15). Characteristic of the skin is the occurrence of type VII collagen in the dermo epidermal basement membrane and also around hair follicles and ducts of eccrine glands, though not around acini of sebaceous or sweat glands (Fig. 16). Using immuno electron microscopy, type VII collagen has been localized at the anchoring fibrils con necting the basement membrane with the interstitial collagen fibers (BuRGESON et a1. 1985; SAKAI et a1. 1986). Although not part of the basement membrane proper, tenascin is of interest here because of its expression in connection with epidermal neoplasms. This extracellular matrix molecule occurs exclusively in blood vessels and in nerve sheaths of normal skin (CHIQUET-EHRISMANN 1986). 5.2.1 Benign and preneoplastic lesions

In benign proliferative lesions of the epidermis, including sebaceous epithelioma, verruca vulgaris and keratoacanthoma, the epidermal basement membrane when stained for type IV collagen or laminin appears to be regular and continuous (Fig. 1). Excep tions to this rule may occur in areas of inflammation, where inflammatory cells invading the epithelium cause circumscript defects in the basement membrane. Type VII collagen distribution has not been studied in these lesions. In solar (actinic) keratosis variations in the thickness of the basement membrane can be observed. However, usually the basement membrane remains intact (GUSTERSON et a1. 1984, 1986; CAM et a1. 1984). A similar pattern can be observed in Bowen's disease; but in association with a reaction in the dermal stroma, including an inflammatory infiltrate, gaps and also duplications in the basement membrane may occur (STENBACK and WASENIUS 1985; GUSTERSON et a1. 1984, 1986). Whether or not these represent areas of incipient invasive growth is unknown. In any case, it would be difficult to distinguish genuine gaps due to early invasion from those elicited by inflammatory reaction. 5.2.2 Epidermoid carcinoma

In epidermoid (squamous cell) carcinoma, a well-defined basement membrane can be observed around the tumor cell nests regardless of the degree of differentiation of the tumor (GuSTERSON et a1. 1984) but usually associated with foci of discontinuity (Fig. 17A, B). STENBACK and WASAENIUS (1985) observed a relationship between basement membrane deposition and degree of differentiation. Thin and partly discontinuous basement membrane surrounded moderately differentiated carcinomas, but irregular basement membranes were found in the center of poorly differentiated carcinomas, without any basement membrane deposition at the invading tumor periphery occurring. In contrast, BARSKY et a1. (1983) reported mostly the absence of basement membrane immunoreactivity in a majority of invasive carcinomas, with only occasionally foci of


Fig. 17. Basement membrane patterns in epidermoid carcinoma (A = 100, B = 250) and basal cell epithelioma (C = x 100, D = x 250) of the skin. Note discrete discontinuities in A and Band ~ontinuous staining in C and D. - Type IV collagen immunoperoxidase staining.

:ype IV collagen and laminin staining occurring in well-differentiated carcinomas. Gus rERSON et a1. (1986) observed duplications and intradermal projections of the basement nembrane in squamous cell carcinomas and basal cell epitheliomas, these being often in :umor areas invaded by inflammatory cells. It was suggested that these basement mem Jrane structures might be remnants of areas of tumor regression. ;.2.3 Basal cell epithelioma

Basement membrane immunohistochemistry in basal cell epithelioma is of special nterest due to the peculiar clinical and histological characteristics of this neoplasm in


relation to its biological behavior. Most cases follow a benign clinical course, but some show deep local invasive growth and a tendency for recurrence. Metastasis seldom occurs though. The desmoplastic or morphea type basal cell epithelioma, a histological ly defined subtype, tends to invade more aggressively than other varieties of this neo plasm. Many studies have focussed on basement membrane immunohistochemistry in basal cell epithelioma and indeed these mostly yield identifical findings: continuous and regular basement membranes lining the tumor nests in nodular and cystic basal cell epithelioma (Fig. 17 C,D) often with patches of immunoreactive material in the center of the nests of tumor cells (CAM et al. 1974; MEROT et al. 1984; GRIMWOOD et al. 1984; GUSTERSON et al. 1984; KALLIOINEN et al. 1984; BASSET SEGUIN et al. 1989). The intact peritumoral basement membrane demonstrates that invasive growth is not inevitably associated with loss of basement membrane staining though it has led some investigators to conclude that most basal cell epitheliomas are not invasive neoplasms. STAMP (1989) recently reported that in basal cell epithelium tenascin is deposited in the peritumoral stroma of basal cell epithelioma. This finding is of interest in view of the expression of tenascin during skin development. Special attention has been paid to desmoplastic basal cell epithelioma (KALLIOINEN et al. 1984b). In contrast to the nodular type this variant shows extensive irregularities and basement membrane defects which sometimes extend to complete absence. This distinc tive pattern is in agreement with the more invasive tendency of this subtype, and also corresponds with the high activity of type IV collagenase found in this subtype as compared with the nodular type. MEROT et al. (1984) studied the localization of bullous pemphigoid antigen (BPA) in basal cell epitheliomas. Contrasting with the continuous and linear immunoreactivity for type IV collagen and laminin exhibited at the periphery of the tumor cell nests, BPA immunoreactivity did not occur at the perimorallacunae which are, in any case, regarded by many as artifacts due to the fixation procedure. This finding was taken to indicate defects in basal cell adhesion at the site of the lacunae. 5.2.4 Nevomelanocytic lesions

In pigmented skin lesions the distinction between naevocellular naevi, dysplasdc naevi, juvenile melanoma and malignant melanoma may create difficulties. In this area, basement membrane immunohistochemistry has provided a useful diagnostic tool and has also provided some insight into complex interactions between tumor cells and reactive stromal elements. Immunohistochemical localization of type IV collagen and laminin in intraepidermal naevi has demonstrated the presence of an intact basement membrane surrounding the nest of naevus cells. Ultrastructurally the lamina densa was found to be thinner than usual for the epidermis, while immunoreactivity for type IV collagen was lacking (SCHMOECKEL et al. 1989). In compound naevi, the dermal cell nests appear to be surrounded by a continuous


basement membrane exhibiting immunoreactivity for type IV collagen and laminin but not for type VII collagen. Ultrastructurally, anchoring fibers are lacking. In areas with an inflammatory infiltrate focal basement membrane discontinuities occur (HAVENITH et al. 1989). This phenomenon occurs especially in halonaevi located at the site of the abundant inflammatory infiltrate, where basement membrane components often cannot be demonstrated. Occasionally basement membrane remnants are found to occur which may indicate tumor regression (GUSTERSON et al. 1984; HAVENITH et al. 1989). In juvenile melanomas, basement membrane characteristics are comparable to those in cellular nevi (HAVENITH et al. 1989; SCHMOECKEL et al. 1989). In some lesions, hyaline bodies occur at the dermal-epidermal junction which are immunoreactive for type VII collagen and laminin (STENBACK and WASENIUS 1986; HAVENITH et al. 1989, SCHMOECKEL et al. 1990). This phenomenon may be of some help in discriminating between juvenile melanoma and malignant melanoma. In view of the fact that malignant melanomas are invasive, several investigators have looked for basement membrane de fects in these lesions, using antibodies to type IV collagen and laminin. Strikingly, even in highly malignant melanoma, in intravascular tumor cell nests and in lymphnode metastases, the tumor cell nests were invariably found to be demarcated by basement membrane material (SCHMOECKEL et al. 1989). In the lentigo maligna melanoma an intact basement membrane invariably occurs, except in areas with an inflammatory reaction (HAVENITH et al. 1989; STENBACK and WASENIUS 1986, 1989). In superficial spreading melanoma, intercellular basement membrane immunoreactivity occurs in the tumor cell clusters at the dermo-epidermal junction and also pericellularly around large clusters of tumor cells (STENBACK and WASENIUS 1986; HAVENITH et al. 1989). Discon tinuities frequently occur in the absence of inflammatory cells, especially in areas of active invasion. In nodular malignant melanoma, inter- and pericellular type IV collagen and laminin immunoreactivity can often be seen around tumor cells adjacent to small vessels. Fre quently basement membrane discontinuities are found to occur (HAVENITH et al. 1989; STENBACK and WASENIUS 1986). Alterations in the composition of the basement mem brane have been reported (SCHMOECKEL et al. 1989). These investigators found almost [10 type IV collagen and nidogen deposition surrounding tumor cell nests in the dermis, but there was abundant intercellular laminin staining. Furthermore type VII collagen was completely absent. These observations suggest that the basement membrane gradu ally disappears in the initial phase of dermal infiltration. KIRKHAM et al. (1989) studied :he localization of type VII collagen. Around lesions less than 0.6 mm thick a basement nembrane was found that exhibited immunoreactivity for type IV collagen, laminin and :ype VII collagen. In lesions thicker than 0.6 mm type VII collagen immunoreactivity :lisappeared. It is tempting to speculate that this reflects loss of tumor cell adherence to ;tromal elements in conjunction with more aggressive behavior in terms of invasion and netastasis. This would be in agreement with clinical data regarding the prognostic ;ignificance of melanoma thickness (BRESLOW 1970).


5.2.5 Cutaneous adnexal tumours KALLIOINEN et a1. (1985) studied cutaneous adnexal tumors by applying type IV collagen and laminin immunohistochemistry. In general, a regular and continuous base ment membrane occurred around the tumor cell nests in benign tumors including apocrine hidrocystoma, nodular and papular hidradenoma, sebaceous adenoma and eccrine spiradenoma. Irregularities (e.g. variation in thickness multiplication and dis continuities) were found in some benign tumors such as trichoepithelioma, pilomatrixo ma and trichofolliculoma. Syringomas displayed a continuous basement membrane around well organized tubular structures, but granular basement membrane depositions were noted surrounding unorganized cell nests. The malignant adnexal tumors showed discontinuous and often irregular deposition of basement membrane material, which is in agreement with the findings for other types of epithelial malignancies. In cylindromas (adenoid cystic carcinomas) abundant basement membrane material is found to have a linear multilayered pattern around tumor cell clusters and in a granular form inside the clusters. Typical extracellular hyaline bodies and extremely thick basement membranes are regularly found (WEBER 1964, 1984; KALLIOINEN et a1. 1984, 1985; BARSKY et a1. 1987).

5.3 The breast 5.3.1 The normal breast In the normal breast the basement membrane configuration has been studied exten sively. A regular and continous membrane surrounding ducts and lobules is found when breast tissue is stained for type IV collagen and laminin. Furthermore blood vessels and lymphatics are stained (ALBRECHTSEN et a1. 1981; BETTELHEIM 1984; BIREMBAUT et a1. 1984; NATALI et a1. 1984; CHARPIN et a1. 1986, 1989; WILLEBRAND et a1. 1986). Cyclic changes may be envisioned for the breast during the menstrual cycle, but this has not been studied as yet. It is also conceivable that during pregnancy and lactation consider able amounts of basement membrane material are deposited and then degraded during post-latational evolution, but again this has not been extensively studied. Other basement membrane components have also been investigated. Fibronectin occurs in basement membranes and also in the connective tissue stroma (BIREMBAUT et a1. 1984; NATALI et a1. 1984; CHRISTENSEN et a1. 1985). Recently, the immunoreactivity patterns for type VII collagen have been described in normal breast tissue (WETZELS 1989). In vascular structures collagen type VII was not found. In glandular structures the staining pattern corresponded with that for type IV collagen and laminin. However, the staining was granular rather than linear. This could be due to the occurrence of type VII collagen in the anchoring fibrils, which do not form a linear structure.


Fig. 18. Type IV collagen immunoreactive basement membranes in fibrocystic disease of the breast. Note basement membrane thickening and multiplication around ducts (A = x 250); acini of sclerosing adenosis infiltrating in the perineural sheath surrounded by a basement membrane (B = x 160); clusters of tubules with epitheliosis surrounded by a continous basement membrane (C = x 160); intraductal papillomatosis with basement membrane containing fibrovascular stalks (D = 100).

5.3.2 Benign proliferative breast lesions

In benign lesions of the breast, including fibroadenoma, fibrocystic disease and gy necomasty, the presence of type IV collagen, laminin and heparan sulphate proteogly :an identify a regular and continuous basement membrane with all epithelial structures :lutlined (CAM et a1. 1984; GUSTERSON et a1. 1984; BIREMBAUT et a1. 1985; CHARPIN et 11. 1986, 1989; WILLEBRAND et a1. 1986). NATALI et a1. (1984) described basement


membrane thickening and splitting in fibroadenoma and in fibrocystic disease. In cases of fibrocystic disease involving epitheliosis, the intraductal epithelial proliferations also display a regular basement membrane (Fig. 18C). In ductal papillomas the central stromal stalk is demarcated by a basement membrane (WILLEBRAND et a1. 1986). This phenomenon can be used to distinguish ductal epitheliosis from ductal papillomatosis (Fig. 18D). Fibronectin immunohistochemistry presents a similar pattern but is found additionally in the adjacent stroma (BIREMBAUT et a1. 1984; NATALI et a1. 1982; BIREM BAUT et a1. 1985; CHRISTENSEN et a1. 1984). Somewhat different results have been reponed in cases of adenosis. In these lesions patches of type IV collagen and laminin immunoreactivity can be found in the intralobular stroma, in addition to the usual delineation of the epithelial structures (GUSTERSON et a1. 1984). This phenomenon is encountered most strikingly in sclerosing adenosis and in radial scar lesions (WILLE BRAND et a1. 1986). The usually desmoplastic stroma found in these lesions contain many myofibroblasts (GUSTERSON et a1. 1982). It is likely that these cells are responsible for the deposition of the basement membrane material in the intracellular stroma. Areas of intraneural invasion in sclerosing adenosis, which are found occasionally, are always surrounded by an intact basement membrane (Fig. 18B). This consistent finding is useful in distinguishing sclerosing adenosis from tubular carcinoma (BARSKY et a1. 1983; EKBLOM et a1. 1984; WILLEBRAND et a1. 1986). 5.3.3 Breast carcinoma

A problem area in breast pathology which could benefit from basement membrane immunohistochemistry is differentiating between intraduct and invasive carcinoma. Furthermore, immunohistochemistry is helpful in the differential diagnosis of tubular carcinoma and lesions such as sclerosing adenosis and microglandular adenosis. In traduct carcinomas have been studied extensively. In areas of intraduct and intralobular carcinoma, regular and continuous basement membranes occur when immunohis tochemistry is performed in the localization of laminin and collagen types IV and VII (SIEGEL et a1. 1981; BARSKY et a1. 1983; LAGACE et a1. 1985; WILLEBRAND et a1. 1986; WETZELS et a1. 1989). Some authors reponed irregular, partly thickened basement membranes (CHARPIN et a1. 1986, 1989) and others state that focal disruptions occur in areas of microinvasion (PAGES et a1. 1984; BIREMBAUT et a1. 1984, 1985). GUSTERSON et a1. (1982) studied basement membrane patterns in relation to the presence or absence of myoepithelial cells, which might playa role in the deposition of ductal basement memb ranes. A close correlation was found between absence of myoepithelial cells and absence of basement membranes in areas of invasive growth. With regard to ductal and lobular invasive carcinoma, there appears to be general agreement that the basement membrane is either lacking or fragmented in areas of invasive growth, with type IV collagen and laminin also presenting a similar pattern. The amount of basement membrane material, however, may vary considerably from


Fig. 19. Basement membrane patterns in carcinoma of the breast. In tubular carcinoma neoplastic tubules are completely devoid of basement membranes (A). Note well delineated capillaries (C). In infiltrative duct carcinoma (B) discontinuous basement membranes (arrows) surround neoplas tic tubules. - Type IV collagen immunohistochemistry (A = x 100; B = x 250).

cumor to tumor. Staining patterns, varying from focal areas of interruption to complete lbsence of a basement membrane, were reported by various authors (NATALI et al. 1984; BIREMBAUT et al. 1985). The data presented by WILLEBRAND et al. (1986) are ;haracteristic in that basement membrane immunoreactivity did not occur in half of the lnvestigated ductal invasive carcinomas, while the other cases displayed fragmented and ;cattered basement membrane components (Fig. 19). Consistent differences in basement nembrane patterns between various subtypes of breast carcinomas were not found. Some investigators reported a correlation between degree of differentiation of the :umor and the extent of basement membrane deposition, with poorly differentiated or maplastic tumors exhibiting almost no immunoreactivity and well differentiated tumors ~xhibiting more extensive basement membrane deposition (BIREMBAUT et al. 1984). ieveral investigators have studied type VII collagen localization in breast cancer. A ~eneral advantage of this basement membrane component appears to be that it does not )Ccur in the vascular wall. The pattern of type VII collagen distribution is not identical ;vith that of type IV collagen and laminin. As has been pointed out previously, it is :onceivable that type VII collagen plays a role in tumor invasion but this has not yet Jeen completely elucidated (WETZELS et al. 1989). 5.4 Respiratory tract The upper respiratory tract mucosa, which is lined with stratified squamous epithelium and with scattered mucous glands in the subepithelial connective tissue,


features a regular and continuous basement membrane. The entire basement membrane contains type IV collagen and laminin (CAM et a1. 1984; GUSTERSON et a1. 1984; SAKR et a1. 1987). The basement membrane of the squamous and ciliated nasopharynx and larynx epithelium contains in addition type VII collagen (unpublished observations). In the lower respiratory tract, including the tracheobronchial tree and alveoli, the lining epithelium is demarcated by a continuous basement membrane. In areas with mucosa associated lymphoid tissue focal interruptions occur, e.g. in Peyer's patches in the gut. 5.4.1 Upper respiratory tract tumors In benign lesions in the nasopharynx and larynx, such as papilloma and inverted papilloma, a regular and continuous basement membrane is invariably found. A peculiar lesion in this region is the juvenile nasopharyngeal angiofibroma. In this lesion the vascular structures display the usual basement membrane but, in addition, deposition of basement membrane material can be found in the fibrous tissue, underlining the myo fibroblastic nature of these cells. VISSER et a1. (1986) have extensively studied hyperplasia, dysplasia and (in situ) carcinoma of the larynx. In hyperplasia and dysplasia of the laryngeal mucosa a regular and continuous basement membrane is found. In carcinoma in situ, some lesions show a continuous basement membrane whereas in others focal interruptions are noted. In invasive squamous cell carcinomas focal interruption of the basement membrane invari ably occur (Fig. 20). Focal basement membrane interruptions in carcinoma in situ possibly identify areas of early (or incipient) invasive growth. This assumption, howev er, needs to be verified by additional studies including clinical follow-up work with patients. A problem in the diagnostic use of basement membrane immunohistochemis try is the occurrence of basement membrane defects in areas exhibiting an inflammatory reaction (Fig. 21), irrespective of the nature of the associated epithelial proliferation (CAM et a1. 1984 a,b; GUSTERSON et a1. 1984; BIREMBAUT et a1. 1985; VISSER et a1. 1986; SAKR et a1. 1987). 5.4.2 Lower respiratory tract Basement membranes in bronchial and lung cancer have been the object of intensive ultrastructural study by DINGEMANS and Moor (1986, 1987) and HAVENITH et a1. (1990) and also immunohistochemically by TEN VELDE (1991). Electromicroscopy confirmed by immunohistochemistry reveals basement membrane patterns in squamous cell car cinoma of the lung as somewhat unusual in appearance, presumably this being related to the pattern of invasive growth which is at least partly dictated by the alveolar architec ture of the lung. In the periphery of the tumors, almost invariably the pre-existing alveolar pattern is found to remain, while the alveolar lumina appear filled with the


~ig. 21. Basement membrane defects in areas of inflammation in a larynx biopsy. - Type IV :ollagen immunohistochemistry (x 250).


Fig. 22. Squamous cell carcinoma of the lung, growing in preexisting alveoli which display an intact architecture. - Type IV collagen immunohistochemistry (A = x 80, B = x 160).

expansively growing tumor cells (Fig. 22). In the center of the tumors the alveolar architecture is obscured and a variable amount of basement membrane material is deposited around tumor cell nest which, at that site, appear to invade into the interstitial compartment and elicit a variable desmoplastic reaction. TEN VELDE et al. (1991) com pared the extent of basement membrane deposition in the tumor center with the biolog ical behavior of the tumor in terms of patient survival. Patients with a tumor containing extensive basement membrane deposits survived significantly longer than those without this characteristic. In lung adenocarcinomas a similar pattern can be found: on the one hand, there is often a remarkably intact alveolar architecture within the periphery; on the other hand, the tumor center exhibits a variable degree of pericellular basement membrane deposition. In neuroendocrine neoplasms of the respiratory tract, the findings vary according to the degree of differentiation of the neoplasm. Well differentiated neuroendocrine car cinomas - conventionally classified as carcinoid (GOULD et al. 1983) consist of regular nests or cords of monomorphic cells invariably surrounded by a regular and continuous basement membrane. Moderately differentiated neuroendocrine carcinomas - conven tionally classified as atypical or spindle cell carcinoids - show a more irregular growth pattern and cytonuclear atypia. In these neoplasms, the basement membranes surround ing tumor cell nests frequently display interruptions. In poorly differentiated neuroen

Basement membranes in neoplasia' 47

docrine neoplasms - commonly classified as small cell carcinoma - the tumor cell nests infrequently show some deposition of basement membrane material. In this category of tumors, therefore, the extent of basement membrane deposition can be said to decrease along with decreasing degree of differentiation.

5.5 The digestive tract Due to the size and complexity of the organ system, the wide spectrum of different tumor types known and their relatively frequent occurrence, neoplasms of the digestive tract have been extensively studied for basement membrane alterations. In view of the rather unique characteristics of the neoplasms in the salivary glands, these organs will be discussed separately as will the pancreas and the hepatobiliary system. It will be be convenient to discuss the mucosal organs together. 5.5.1 Salivary glands

Ducts and acini in the normal salivary glands are outlined by a regular continuous basement membrane, containing laminin and type IV collagen (TOIDA et al. 1984). In salivary gland tumors characteristic alterations of this extracellular matrix compartment have been found. In pleomorphic adenomas a variety of growth patterns occurs, including myxomat ous, chondromatous, solid and tubular formations (ERLANDSON et al. 1984). In the myxomatous areas discontinuous fragments of basement membrane material can be found that are reactive to laminin and type IV collagen. Chondromatous areas do not contain any basement membrane components. In solid cell nests small linear deposits of laminin and type IV collagen can be found extracellularly on occasion. Tubular areas, however, are usually outlined by distinct continuous basement membranes (TOIDA et al. 1984). Fairly characteristic patterns of basement membrane deposition have been noted in adenoid cystic carcinoma, in the salivary gland, and also in other sites (TOIDA et al. 1984, 1985; WEBER et al. 1984; KALLIOINEN et al. 1985; CASELITZ et al. 1986; BARSKY et al. 1987). A striking histological landmark in adenoid cystic carcinoma is the deposition of sheaths of dense amorphous eosinophilic material contiguous to the cylindrical cell clusters. In the lumen of the cylinders this material occurs as hyaline bodies. Electron microscopy has shown this material to consist of reduplicated basement membrane material. Immunohistochemistry reveals dense depositis of type IV collagen (Fig. 8), frequently exhibiting a multilayered pattern. A thin and occasionally discontinuous basement membrane is usually detected around trabecular cell formations. In the tumor cells, especially those adjacent to the basement membrane, cytoplasmic deposits of type IV collagen and laminin can often be discerned (Fig. 8). The ample presence of basement membrane components in this tumor has been regarded as evidence suggesting the


myoepithelial origin of most salivary gland tumors (CASELITZ et al. 1986). Patterns of basement membranes in other salivary gland tumors are less characteristic. In the (be nign) Warthin tumor the ducts and cysts are outlined by a regular continuous basement membrane. In mucoepidermoid tumors, regular tubular formations tend to be sur rounded by a regular basement membrane, though discontinuities frequently occur. In adenocarcinomas irregular and discontinuous basement membranes usually prevail. In spite of the striking patterns in pleomorphic adenoma and adenoid cystic carcinoma, there is little diagnostic use made of basement membrane immunohistochemistry in these neoplasms. Basement membrane patterns are not informative in respect of recurr ence potential (pleomorphic adenoma) or malignancy grade (adenoid cystic carcinoma). 5.5.2 Oral tissue and esophagus

The oral and esophageal mucosa is lined by a non-keratinizing squamous epithelium, resting on a regular and continuous basement membrane. In the anchoring fibrils this contains type IV collagen and laminin as well as type VII collagen. The basement membrane remains intact in hyperplastic and dysplastic lesions of the squamous epithe lium. The basement membrane appears to be interrupted in squamous cell carcinoma (MEYER et al. 1985). Of the odontogenic tissue, the odontogenic epithelium and the epithelium of the enamel apparatus both contain a well developed basement membrane consisting of type IV collagen, laminin and type VII collagen. The latter, however, does not occur in the enamel epithelium. Adjacent to the stromal side of the basement membrane is located an abundant layer of tenascin immunoreactive material. In odontomas, ameloblastomas and ameloblastic fibromas, the tumor epithelium is demarcated by a regular continuous basement membrane containing type IV collagen, type VII collagen and laminin (THEs LEFF et al. 1981, 1984, 1987; HEIKINHEIMO et al. 1991). Focal interruptions of the basement membrane have been noted in ameloblastomas (SAUK et al. 1985). Further more, tenascin was noted in all tumors of odontogenic tissues (HEIKINHEIMO et al. 1991 ). 5.3.3 Stomach, small and large bowel

Although important functional and structural differences exist between the glandular mucosae along the gastrointestinal tract from the point of the view of the basement membrane, they are rather uniform. The epithelial lining of the gastrointestinal tract is demarcated by a continuous basement membrane exhibiting a lamina rara and a lamina densa. The composition of the basement membranes as usual features type IV collagen and laminin as the dominant molecular components. Anchoring fibrils are lacking and hence type VII collagen is not found in the normal gastrointestinal tract, with the exception of the esophageal and anal mucosa. A characteristic feature of the basement


membrane in the gastrointestinal tract is the occurrence of discontinuities or fenestra tions overlaying lymphoid follicles in the lamina propria. This feature is presumably related to the necessity for antigen presenting cells to pass through the epithelial lining. Neoplastic lesions of the stomach and the small intestines have already been studied with regard to basement membrane characteristics. Our own observations indicate that in non-neoplastic lesions, such as hyperplastic polyps and hamartomatous polyps, in tact basement membranes underlie the glandular structures. Under dysplastic condi tions, including dysplasia in gastric type mucosa in Barret's oesophagus and in gastroje junostomy mucosa, intact basement membranes usually line the distorted glands, al though in areas with active inflammation circumscript defects are found to occur where inflammatory cells invade the epithelium. In contrast, extensive defects occur in adenocarcinomas in the absence of inflammatory cells. In poorly differentiated lesions, such as diffuse type (signet ring cell) carcinoma, basement membranes are almost com pletely lacking. A striking basement membrane pattern is seen in neuroendocrine tumors in the stomach, small bowel and appendix. Almost invariably the tumor cell nests in car cinoids are surrounded by a well developed continuous basement membrane, regardless of whether or not the lesion possesses malignant potential (Fig. 23). Only in so-called

Fig.23. Patterns of basement membrane deposition in an appendiceal carcinoid. The tumor is poorly demarcated but nests of tumor cells are surrounded by a regular basement membrane. Type IV collagen immunohistochemistry (x 100).


signet ring cell carcinoids (which occur most frequently in the appendix and tend to invade diffusely into the bowel wall) may basement membrane deposits be lacking. Basement membrane configurations have been studied rather extensively in the colon (BURTIN et al. 1983; FORSTER et al. 1984, 1986; DANEKER et al. 1987; HAVENITH et al. 1988). BURTIN et al. (1983) were the first to report that, in colonic adenomas, in general, the tumor glands are demarcated by a regular continuous basement membrane, contain ing laminin as well as type IV collagen and invariably codistributed. In adenomas exhibiting areas of severe dysplasia, focal interruptions of the basement membrane may occur, which is suggestive of progression of the lesion towards an invasive adenocar cinoma (BOSMAN et al. 1985). In adenocarcinomas it is usually found that areas of interruption of the basement membranes occur, which is indicative of the invasive character of the neoplasm (Fig. 24). It is striking, however, that in cases of lymph node or liver metastases of colonic adenocarcinomas, well developed basement membranes frequently occur, especially if the neoplasm is well differentiated. In a study rather similar to the work on laminin immunolocalization as reported by FORSTER et al. (1984,1986), HAVENITH et al. (1988) studied type IV collagen deposition in colorectal carcinoma. Both groups reported a positive correlation between the extent of basement membrane deposition, as reflected in either type IV collagen or laminin immunoreactivity and also in tumor prognosis. In the study of FORSTER et al. (1986) a patient with a laminin positive adenocarcinoma had a 2.7 times better chance of surviv-

Fig. 24. Basement membrane interruptions (arrows) in colonic adenocarcinoma. - Type IV colla gen immunohistochemistry (x 250).


ing 5 years than a laminin negative carcinoma. HAVENITH et al. (1988) noted that the basement membranes were deposited especially in the tumor center, which would fit in with tumor invasion occurring mainly in the tumor periphery. A tendency was noted for basement membrane deposition to be correlated with a desmoplastic reaction. Furthermore, both authors indicate that basement membrane deposition is correlated with differentiation, with highly differentiated tumors displaying more basement mem brane material than poorly differentiated tumors. In a recent study, VISSER et al. (1991) drew attention to alterations in the composition of the basement membrane in colorectal neoplasia. In normal colonic mucosa, type VII collagen does not occur in epithelial basement membranes. In conditions of dysplasia, including dysplasia in long-standing ulcerative colitis and in colonic adenomas, type VII was found to occur, especially at the luminal zone of the dysplastic epithelium. In cases of carcinoma, type VII collagen deposition again disappeared except in the bordering so-called transitional mucosa, surrounding the lesion (Fig. 25). The significance of these findings is a yet unclear but they suggest that type VII collagen, which in a normal basement membrane is localized in anchoring fibrils, is somewhat involved in the transi tion from a premalignant to a malignant condition, as has also been reported for cutane ous malignancies (KIRKHAM et al. 1988 a,b).

~ig. 25. Type IV (A) and VII (B) collagen deposition in transitional mucosa bordering a tubular Idenoma in the colon. Immunoreactivity for type IV collagen is homogeneous but for type VII :ollagen somewhat granular and most intense bordering the lumen. - Immunofluorescence x 250).


An interesting question is how these differences in the deposition of basement mem brane components occur. Here an important consideration is that the observed base ment membranes represent the net balance between deposition of newly synthesized components and degradation of existing components. Hence, absence of basement membranes could indicate high activity on the part of basement membrane degrading enzymes (e.g. type IV specific collagenases, cathepsins) or else inability on the part of tumor cells to synthesize type IV collagen and laminin or both. It has been demons trated that type IV collagen specific collagenases are synthesized in highly invasive neoplasms, which indicates that basement membrane degradation certainly plays a role (LIOTTA et al. 1983; BARSKY et al. 1983). DANEKER et al. (1987) studied the rate of synthesis of laminin in poorly and well differentiated colorectal carcinoma cells and found similar levels of synthesis, though marked differences occurred in the ability to deposit laminin extracellulary. Poorly differentiated carcinoma cells secreted less lami nin, this being presumably due to aberrant posttranslational processing. CLEUTJENS et al. (1990) demonstrated that type IV collagen in basement membranes of colorectal carcinomas is at least partly deposited by stromal cells and that cancer cells differ markedly in their ability to synthesize type IV collagen. Taken together, these findings indicate that absence of basement membranes in a carcinoma reflects inability to synth esize or secrete the components, a high propensity to degrade basement membranes and an insufficient host response. This combination of events is then responsible for the aggressive behavior of the tumor. 5.5.4 The pancreas

The ducts and acini of the normal pancreas are demarcated by a regular and continu ous basement membrane. The individual cells nests and trabeculi in islets are all sur rounded by a disrete basement membrane. Basement membrane alterations have not been studied extensively in pancreatic neo plasms. In their study of a variety of neoplasms BARSKY et al. (1983) mention basement membrane discontinuity occurring in pancreatic adenocarcinoma. HAGLUND et al. (1984) studied basement membranes using laminin immunohistochemistry. In chronic pancreatitis there was some thinning of the basement membrane but no discontinuity was noted. In adenocarcinomas, however, discontinuities were observed. In poorly differentiated carcinomas basement membranes were completely absent but occasional ly intracellular laminin immunoreactivity was seen. Mucinous cystic neoplasms of the pancreas always showed an intact basement membrane, regardless of their behavior. Islet cells tumors also showed a continuous basement membrane surrounding the nodu lar, trabecular or acinar cell nests, regardless of the malignant potential (Fig. 26). HABERERN-BLOOD et al. (1987) studied in vitro laminin expression in different hu man pancreatic adenocarcinoma cell lines; tumors formed by xenografting these cell lines in nude mice showed a tendency to deposit a basement membrane. Their findings


Fig. 26. Islet cell tumor of the pancreas, stained for type IV collagen. The pattern of basement membrane deposition is quite comparable to that in Fig. 23. - (x 100).

ndicate, as in the case of colorectal carcinoma, that the extent of basement membrane oss in pancreatic carcinomas is not due alone to the inability of the tumor cells to ;ynthesize laminin. Basement membrane degrading capacity and the host stromal re ;ponse are also involved. ;.5.5 The hepatobiliary system

In the normal liver, striking differences exist between hepatocytes and biliary with regard to the epithelial basement membrane. In the portal triads, the Jile ducts are demarcated by a regular and continuous basement membrane. In scleros ng cholangitis HAGLUND et al. (1989) noted a somewhat irregular but mostly continu JUS basement membrane by laminin immunohistochemistry. In sclerosing cholan ~iocarcinoma a heterogenous situation was encountered. Largely intact basement nembranes were seen in areas of high differentiation. However, in less well differenti lted areas extensive interruption, or even complete absence of, the basement membrane ;vas invariably noted. Basement membrane immunohistochemistry can therefore be lsed to differentiate between these conditions, which can be easily confused by regular listological examination. In the liver parenchyma the situation is quite different. Hepatocytes do not rest upon l continuous basement membrane. The sinusoidal lining, consisting of fenestrated en iothelial cells, does not rest on a continuous basement membrane. Rather irregular ~pithelium

S4 . F. T. Bosman et al.

patches of type IV collagen and fibronectin immunoreactive material can be observed (HAHN et al. 1980; MARTINEZ-HERNANDEZ et al. 1984, 1985). Laminin, however, ap pears to be absent (BIANCHI et al. 1984; DONATO et al. 1989). Contrasting with the absence of a basement membrane adjacent to normal hepatocytes, in hepatocellular carcinomas it is usually found that fragmented basement membranes surround the nodular trabecular or acinar tumor cell groups. As in many other carcinomas, the degree of differentiation appears to be reflected in the tendency to basement membrane deposi tion, with well differentiated tumors showing more extensive basement membranes than poorly differentiated tumors (DONATO et al. 1989). Contrasting with the situation in the normal liver, in hepatocellular carcinomas the basement membrane fragments do contain laminin (TARBARIN et al. 1987; DONATO et al. 1989). 5.6 Endocrine system

The general tissue architecture of endocrine organs is remarkably constant, regardless of the specific tissue architectural features of the various organs. Characteristically it is found that functionally active cells are arranged in small nests; here single layered sheets or bilayered trabeculae are characterized by very little extracellular matrix, but do exhibit a prominent capillary network in close proximity to the parenchymal cells. In all organs, the functionally active cell clusters are demarcated by a regular continuous basement membrane. Fusion of this basement membrane with that of an adjacent capil lary can occasionally be found, yielding a trilaminar structure such as in the renal glomerulus. Based on immunohistochemistry, the basement membrane in endocrine organs contains type IV collagen, laminin and heparan sulphate proteoglycan. The basement membrane in endocrine organs presumably has an important function in allowing hormonal products to gain access to the circulation. In relation to this func tion, it is conceivable that fenestrations occur in these basement membranes. In the tumor pathology of the endocrine system, one important problem appears to be common to all organs of this system. Nodular enlargement of the organs frequently occurs. In some organs, including the pituitary, this almost invariably involves a benign adenoma. In other organs, including the thyroid, the adrenals and the parathyroids, a spectrum of lesions can be encountered varying from nodular hyperplasia, via adenoma to carcinoma. It has proved rather difficult, if not almost impossible, to define histologi cal criteria to reliably distinguish between these categories that obviously differ consid erably in their biological behavior. Theoretically, basement membrane patterns might have been of help here, at least for the distinction between adenoma and carcinoma. In the literature, however, little attention has been paid to this possibility. As mentioned above, in the thyroid the follicles are demarcated by a regular and continuous basement membrane. This structure remains remarkably constant in various growth disorders of the thyroid. Both in nodular· goiter and in thyroid follicular adenomas the follicles show a normal basement membrane appearance. In some


Fig. 27. Type IV collagen immunoreactivity in follicular carcinoma of the thyroid. Nests of tumor ;ells are surrounded by a continuous basement membrane (open arrows), even in vascular lumina :basement membrane indicated by closed arrows) in an area of angioinvasive growth. - (x 100).

trabecular adenomas the tumor cells show a tendency to deposit increased amounts of Jasement membrane material, leading to an inceased hyalin-like extracellular matrix :KATOH et al. 1989). In follicular carcinomas generally an association is noted between :he degree of anaplasia and the basement membrane pattern. In well differentiated iollicular carcinomas a regular and continuous basement membrane is found, even lround invading tumor cell nodules in vascular lumina (Fig. 27). With increasing ana Jlasia, however, interruptions appear, and in anaplastic carcinoma of the thyroid little Jr no basement membranes are encountered. In papillary carcinomas a regular basement nembrane is also frequently retained (CHARPIN et al. 1985; BIREMBAUT et al. 1985). \1edullary carcinomas exhibit scattered deposits of basement membrane fragments. :;ARBI et al. (1988) investigated the production of basement membrane components in a fifferentiated thyroid epithelial cell line and found high levels of laminin and type IV ;ollagen in vitro. These findings indicate that thyroid carcinoma cells retain the ability :0 synthesize and deposit basement membrane material even in frankly malignant con fitions. For problems in histopathological evaluation of thyroid neoplasms, therefore, )asement membrane immunohistochemistry is of little use. Even less is known about basement membrane patterns in pathological conditions in he other endocrine organs. Our own experience indicates that, in the parathyroids and tdrenals, hyperplastic nodules and adenomas retain the ability to deposit basement nembranes in a regular pattern. In the few cases of parathyroid carcinoma and adrenal :ortical carcinoma we have encountered, a remarkably normal basement membrane )attern was found even in invasive areas of the tumor and in metastatic foci. In pheo


chromocytomas of the adrenal medulla the neoplastic cell nodules (classically called «Zell ballen») are also demarcated by a regular and continuous basement membrane. In pituitary lesions the basement membrane pattern is not different from that in the other endocrine organs. In conditions of hyperplasia and of adenoma regular basement membranes are encountered. LEARDKAMOLKARN et al. (1989) reported laminin biosyn thesis by anterior puititary endocrine cells, which indicates that also in the pituitary, the epithelial cells are at least partly responsible for the deposition of the basement mem brane.

5.7 Genito-urinary tract In contrast to most other parenchymal and mucosal epithelia, most of the genito urinary epithelia are of mesodermal origin. Against this background unique basement membrane characteristics might be expected and, indeed, have been shown to exist. An example is the tendency of the endometrial stromal cells to deposit a basement mem brane, this being exclusively confined to the terminal period of the secretory phase and to the decidua during pregnancy. Other peculiarities will be discussed under the diffe rent headings. 5.7.1 The kidneys

The basement membranes in the kidney have received intense attention though al most exlusively in connection with renal glomerular disease. The tubular basement membrane has not been studied to the same extent (GOOVAERTS et al. 1990). A number of specific aspects have emerged from these studies. First of all, the trilaminar nature of the glomerular basement membrane, which is characteristic but not only specific for renal glomerular basement membranes, should be emphasized (MARTINEZ-HERNANDEZ and AMENTA 1983). This configuration is the result of fusion of the epithelial and endothelial basement membranes, which yields one lamina densa in combination with two laminae rara: the external (epithelial) and the internal (endothelial). Secondly, the high concentration of heparan sulphate proteglycans deserves special attention. This feature is related to the selective permeability of the glomerular basement membrane, not only in relation to molecular size but also to molecular charge: heparan sulphate proteglycans are anions. Thirdly, the occurrence of organ-specific characteristics should be mentioned. This is illustrated by the events occurring in the Goodpasture syndrome, which represents a form of glomerulopathy induced by antibodies against basement membrane components, with symptoms reflecting pulmonary and glomerular damage. This latter finding implies that pulmonary and renal basement membranes must have unique characteristics in common. Recent findings have revealed that the so-called «Goodpasture antigen» is in fact an epitope on the Ne1 domain of the type IV (0)4


collagen chain (HUDSON et al. 1989) which occurs in all basement membranes, but may be «hidden» due to an organ-specific supramolecular configuration of type IV collagen (WIESLANDER et al. 1984; CLEUTJENS et al. 1989). Renal adenocarcinomas have not been extensively studied in regard of basement membrane patterns. Our own (unpublished) investigations have indicated that in well differentiated tubular or papillary carcinomas a well developed basement membrane can be found which is often almost continuous (Fig. 28). In poorly differentiated car cinomas or in anaplastic areas in a well differentiated carcinoma, interruptions in, or the absence of, basement membranes may occur (Fig. 29). In spindle cell (sarcomatous) renal cell carcinoma, basement membranes can be entirely absent. In view of the con troversy concerning the biological behavior of small (less than 3 cm in diameter) tubular renal cortical neoplasms, VISSER et al. (1992) have recently made an extensive study of basement membranes in renal cortical neoplasms in relation to tumor size and nuclear grade. Small cortical tubular neoplasms (with a diameter of less than 3 cm) tend to be regarded as benign, in view of the fact that these lesions rarely metastasize. It was therefore hypothesized that in these small cortical neoplasms the epithelial basement membranes would be intact, in contrast to larger neoplasms which would show a 1iscontinuous basement membrane. The data obtained indicate that some small neo :llasms, mostly those larger than 1 cm in diameter, also exhibit basement membrane interruptions. Lesions of up to 1 cm diameter were almost exclusively of low nuclear ~rade and all these lesions had intact basement membranes. Some lesions of between 1

~ig. 28. Deposition of regular (type IV collagen immunoreactive) basement membranes in well lifferentiated (low grade) renal adenocarcinoma. - (x 160).


Fig. 29. Poorly differentiated renal adenocarcinoma, showing fragmentation and focal absence (arrows) of basement membranes. - Type IV collagen immunohistochemistry (x 100).

and 3 cm in diameter did show basement membrane interruptions, mostly in combina tion with a higher nuclear grade. A correlation with tumor behaviour was not made but these data suggest that basement membrane interruptions in small cortical tubular neo plasms are, in combination with a higher nuclear grade, an indication of malignant potential. 5.7.2 Ureter and bladder

Of the ureters and bladder the epithelial basement membrane has not been specifical ly studied but, in our experience, it has a regular continuous appearance, and the usual components are present. Several investigators (CONN et al. 1987; DAHER et al. 1987; HASHIMOTO and SASHITA 1986; SCHAPERS et al. 1990) have reported on the basement membrane patterns in papillary carcinomas of the bladder. Although papillary carcinomas of the renal pelvis and ureter have not been specifically mentioned in these studies, the histological and biological similarities between these tumors and those in the bladder suggest that the data obtained for the bladder will also hold good for the renal pelvis and ureter. In general, grade I papillary carcinomas have a distinct and continuous basement mem brane. In invasive grade I lesions, the basement membrane shows interruptions and in grade II and III tumors extensive interruptions occur as well as areas marked by com


Fig. 30. Basement membrane patterns in papillary carcinoma of the bladder. In grade I carcinoma ,he papillae are all demarcated by a regular basement membrane (A) whereas in grade II carcino ma interruptions occur (B). - Type IV collagen immunohistochemistry (A = x 40, B = x 250).

plete absence of basement membranes (Fig. 30). SCHAPERS et al. (1990) concluded that basement membrane interruptions do not provide a new and independent parameter for grading of papillary urothelial carcinomas, but that basement membrane immunohis tochemistry does faciliate the recognition of invasion in these tumors and is therefore of practical significance for the histopathologist. In line with this opinion, basement mem brane interruptions have been shown to have prognostic significance regarding the :hances of the neoplasm recurring (DAHER et al. 1987; SCHAPERS et al. 1990). In mul tivariant analysis this parameter appeared to be almost completely accounted for by the bistologically assessed tumor invasion (SCHAPERS et al. 1990). 5.7.3 Male genital tract

Of the basement membranes of prostate, seminal vesicles, testis and epididymis only those in the prostate have been exensively studied. All the ductular elements in the ...arious organs are lined by a regular and continuous basement membrane, which ap pears to contain the usual elements. Two facts deserve to be mentioned. Firstly, in the Jrostate the ductules are surrounded by a layer of myoepithelial cells. These are in mlved in the maintenance of the basement membrane structure; immunoreactivity for


type IV collagen and laminin can be demonstrated in these cells (SINHA et al. 1989). Furthermore, a fairly diffuse distribution of type IV collagen and laminin can be en countered in the prostatic stroma in connection with the smooth muscle component. A second noteworthy fact is that in the testis the basement membranes of the seminiferous tubules display an age related tendency towards thickening, this being most remarkable in testicular atrophy. Immunohistochemically, these thickened basement membranes appear to consist of multilayered deposits of type IV collagen and laminin. As far as neoplastic conditions of the male sex organs are concerned relatively little attention has been paid to the role of the basement membrane. In the case of lesions of the epididymis and seminal vesicles basement membranes have not been studied at all. Basement membranes have been studied in the case of preneoplastic and neoplastic conditions of the prostate and in (testicular) germ cell tumors (ULBRIGHT et al. 1986; SINHA et al. 1989). Hence the discussion will be limited here to these two aspects. As is the case in many other organs, premalignant neoplastic conditions have been identified in the prostate. The nomenclature of these lesions has not been standardized though. Some authors use the term dysplasia (involving a three-step grading system) and others the term prostatic intraepithelial neoplasia (PIN, also involving a three-step grading system [BOSTWICK and BRAWER 1987]). Neither of these systems has been universally adopted and many pathologists are hesitant in recognizing premalignant prostatic lesions. In these lesions - which by definition remain confined to preexisting ductules - the lining with myoepithelial cells can usually be recognized and, in conjunc tion with this finding, the ductular basement membrane is found to remain intact. Only in severe dysplasia or in PIN III, can lesions be found devoid of myoepithelial cells and, in this situation, the basement membrane shows discontinuities. In prostatic cancer desintegration of the basement membrane structure is exhibited. The extent of basement membrane deposition in prostatic cancer tends to run parallel with the degree of dif ferentiation (SINHA et al. 1989). In mostly glandular carcinomas many neoplastic tubu les will be surrounded by a basement membrane that is often continuous. In solid poorly differentiated adenocarcinoma basement membranes are usually scanty or absent (Fig. 31). This illustrates that basement membrane deposition is not solely dependent on the presence of myoepithelial cells in the prostate. Conceivably, the smooth muscle cell rich stroma might playa role in the deposition of basement membranes in the case of prostatic cancer. The practical significance of basement membrane immunohistochemis try in histopathological diagnosis of prostatic neoplasia is limited. The possibility that discontinuities in the basement membrane might reveal incipient invasive growth in premalignant neoplastic lesions is a possibility that should be further evaluated. In testicular tumors little attention has been paid to basement membranes. In investi gations on seminomas it has been recognized that intratubular or in situ seminomas occur. Since neoplastic cells in this lesions are confined within a seminiferous tubule, basement membrane immunohistochemistry might facilitate its recognition. No base ment membrane studies into seminoma have been performed though. With regard to


Fig. 31. Basement membrane staining pattern in prostatic cancer. The stromal cells (5) are almost all surrounded by a basement membrane whereas the neoplastic glands show basement membrane fragments (arrows). - Type IV collagen immunohistochemistry (x 160).

Fig. 32. Type IV collagen immunohistochemistry in decidua. Note basement membrane deposi tion around individual decidual cells. - Immunoperoxidase (x 250).


germ cell tumors some attention has been paid to aspects of the basement membrane (ULBRIGHT et al. 1986), albeit in the wider sense of germ cell tumors generally (includ ing e.g. ovarian germ cell tumors) where experiental models of embryonal carcinoma have mostly been focussed on (MARTINEZ-HERNANDEZ et al. 1982). In fact, early studies on basement membrane morphology and composition were performed on mouse mod els of embryonal carcinoma; it was recognized that in this tumor an equivalent of the parietal endoderm can be found reposing on a basement membrane, the latter being the equivalent of Reichert's membrane in the developing embryo (PIERCE et a1. 1962). Murine yolk sac tumors have been used as a source of the basement membrane compo nents laminin and type IV collagen (LISSITZKY et al. 1983). In human embryonal cell carcinomas type IV collagen and laminin can be readily detected by immunohis tochemistry, especially in yolk sac (or endodermal sinus) components. The hyaline globules, one of the characteristic components of yolk sac tumors, exhibit immunoreac tivity with respect to these basement membrane components (ULBRIGHT et a1. 1986). Although in terms of differentiation of embryonal stem cells and the role played by extracellular matrix components these results are highly interesting, their significance is purely theoretical. In terms of histopathological diagnosis basement membrane Im munohistochemistry does not seem to playa role in gonadal germ cell tumors. 5.7.4 The female genital tract In the female genital tract basement membranes have been studied fairly extensively, especially in conjunction with studies concerning basement membrane alterations in neoplastic conditions of these organs. All epithelial linings of the female internal genitalia are supported by a regular continuous basement membrane. The situation existing in the endometrium is rather characteristic. In proliferative endometrium, base ment membranes are found only in conjunction with the epithelial surface and vascular endothelium. In the secretory phase, however, laminin immunoreactivity has also been found in decidualized stromal cells (FABER et al. 1986). This situation is continued during pregnancy in the decidua, the decidual cells being surrounded by a basement membrane with a typical lamina rara and a lamina densa (Fig. 32). (CHARPIN et al. 1985; WEWER et al. 1985). A relatively large number of reports describe basement membrane characteristics in both benign and malignant conditions of the endometrium (STENBACK et al. 1985; FABER et al. 1986; FURNESS and LAM 1987; VOGEL and MENDELSOHN 1987; BULETTI et al. 1988). These reports generally confirm the existence of an intact epithelial basement membrane in normal mucosa and in cystic hyperplastia of the endometrium. In general, intact basement membranes are also reported in adenomatous hyperplasia of the en dometrium. FURNESS and LAM (1987) reported disruptions of the basement membrane in some cases of adenomatous hyperplasia. The pattern in well differentiated endomet rial adenocarcinoma was by and large similar to well developed basement membranes


Fig. 33. Interruptions of basement membrane type IV collagen staining (arrows) in endometrial adenocarcinoma (x 250).

surrounding neoplastic glands, though there were often some interruptions (Fig. 33). In poorly differentiated endometrial adenocarcinoma, little or no basement membrane deposition was detected though. Interestingly, progestin treatment of adenocarcinomas has been found to induce deposition of epithelial basement membranes (BULETII et al. 1988). Basement membrane components were detected in the endometrial stroma in adenomatous hyperplasia, although less extensively than in normal secretory endomet rium. In endometrial adenocarcinoma the stromal cells did not show a basement mem brane. However, progestin treatment increased the tendency for basement membrane deposition in the stroma of adenomatous hyperplasia as well as in adenocarcinoma (BULLETTI et al. 1988). This finding indicates that the tumor stroma of endometrial adenocarcinomas retains functional characteristics of endometrial stroma. Taken to gether, these reports underline the practical significance of basement membrane his tochemistry for the histopathological diagnosis of neoplastic lesions of the endomet rium. This approach may assist in the differentiation between endometrial hyperplasia and adenocarcinoma and in adenocarcinoma grading. Also, dating of endometrial biop sy specimen in the menstrual cycle can be facilitated on the basis of basement membrane deposition around stromal cells. Several investigations have focussed on basement membrane alterations in lesions of the uterine cervix (STENBACK et al. 1985; VOGEL and MENDELSOHN 1987; EHRMANN et


al. 1988). STENBACK et al. (1985) report that inflammatory lesions in the uterine cervix lead to interruption of the basement membrane of the cervical epithelium, which characteristically is continuous. Continuity appears to be retained in dysplastic condi tions and even in carcinoma in situ, although STENBACK et al. (1985) suggest that in carcinoma in situ focal disruption might occur. In squamous cell carcinoma irregular basement membranes occurred with focal disruptions; but in poorly differentiated carcinoma there was an almost total absence of basement membrane staining. These findings suggest that the differentiation between non-invasive neoplastic conditions of the cervix uteri and invasive carcinoma might be facilitated by basement membrane immunohistochemistry. However, in view of the basement membrane irregularities in inflammatory lesions, this approach should not be used as a sole criterion for malignan cy, as has been shown for the larynx by VISSER et al. (1986). Some attention has been paid to basement membrane patterns in ovarian neoplasms (STENBACK and WASENIUS 1985; FRAPPART et al. 1984). A fairly comprehensive over view of the patterns of basement membrane staining in ovarian neoplasms has been provided by STENBACK and WASENIUS (1985). Benign surface epithelial tumours, e.g. mucinous and serous cystadenomas, displayed a continuous basement membrane sur rounding the epithelial structures. In mucinous and serous cystadenocarcinomas dis ruption of the basement membrane structure was noted, with interruptions occurring around invading epithelial cell nests. We have recently studied borderline lesions of the ovary (unpublished results). On the basis of the basement membrane patterns these tumors can be subdivided into a group with continuous basement membranes, compar able to cystadenomas, and a group with basement membrane interruptions, comparable to cystadenocarcinomas (Fig. 34). Follow-up information on this study was not forth coming and therefore the viability of this criterion for further classification of border line lesions still remains to be validated. According to STENBACK and WASENIUS (1985) carcinosarcomas and mixed Mullerian tumors of the ovary show basement membrane deposition in epithelial areas; but in non-epithelial (spindle cell, sarcomatous or mesen chymal) elements too fragments occurred between the strands of stromal cells, while in fibromas basement membrane material was absent. A distinct and almost continuous basement membrane was found in connection with tubular structures in androblas tomas and also surrounding cell nests in granulosa cell tumors. Immunoreactive base ment membrane material has also been demonstrated in the lumina of tubular structures of granulosa cell tumors. KAUPPILA et al. (1988) suggested that the level of procollagen type III propeptide in the circulation might be used as a prognostic marker for ovarian cancer since it is an indicator of invasive activity. A highly variable pattern of basement membrane staining occurs in germ cell tumors, largely corresponding with that of testicular germ cell tumors. Cell nests in dysger minoma are surrounded by a delicate basement membrane. In embryonal carcinoma a variable amount of basement membrane deposition occurs; it ranges from being frag mented in the tubular configuration to being fairly abundant in yolk sac (endodermal

Basement membranes in neoplasia . 6S

Fig. 34. Basement membrane pattern in borderline tumor of the ovary. In these lesions usually the basement membrane is intact (A) but focally interruptions occur (B, arrows). - Type IV collagen immunohistochemistry (x 160).

sinus) elements. Organoid structures in mature teratomas exhibit regular basement membranes. Taken together, these findings indicate that some diagnostic difficulties in ovarian tumor pathology can at least partly resolved by basement membrane immunohis tochemistry. These include the classification of ovarian borderline lesions and the as signment of poorly differentiated neoplasms to specific categories on the basis of the pattern of basement membrane deposition (FRAPPART et al. 1984).

5.8 Nervous system Basement membranes are not very prominent in the central nervous system, only occurring in the parenchymal tissues of the vascular tree, the arachnoidal lining, the ependymal cells and the choroid plexus (PETERS et al. 1976). Interestingly, laminin production has been reported by early rat astrocytes in culture (LIESI et al. 1983). In the vascular tree basement membrane structure and composition appears to be comparable


to that found in other organs. It is not unlikely, that the basement membrane does have specific characteristic in connection with the specific function of the brain vascular tree in maintaining the blood-brain barrier. This issue has not, however, been completely resolved. In the Schwann cell basement membrane merosin (a laminin related protein) has been described as a rather specific component (LEIVO et al. 1989). Both ependymal cells and the lining cells of the choroid plexus repose on a regular continuous basement membrane, and this holds true also for the arachnoidal (meningothelial) lining cells. In the peripheral nervous system, basement membranes are prominently present around Schwann cells. Basement membrane immunohistochemistry seems to be of practical value for resolv ing various diagnostic difficulties in the tumor pathology of the nervous system (GIOR DANA et al. 1985). Because the vascular tree is prominently stained with antibodies raised against laminin and type IV collagen, a very general application is in the visualiza tion of the general tissue architecture, this being of help in the analysis of the whole spectrum of central nervous system neoplasms. Corresponding with the general pattern for other organs, basement membrane deposition occurs only in tumors consisting of cells that normally are actively engaged in basement membrane production. This implies that basement membrane deposition does not occur in gliomas, with the exception of glioma-sarcomas (MCCOMB and BIGNER 1985) ependymomas and choroid plexus papil lomas. In typical ependymomas the acinar and rosette-like structures are usually out lined by a discrete continuous, basement membrane. But this feature is lost in anaplastic glioblastoma-like ependymomas. Also in the myxopapillary ependymoma of the filum terminale basement membranes are prominent represented in the papillary structures. The papillary structures of choroid plexus papillomas are likewise demarcated by a prominent continuous basement membrane. Basement membrane depositions do not occur in neuronal tumors. Relatively ample attention has been paid to basement membrane patterns in mening iomas (MCCOMB and BIGNER 1985). Arachnoidal lining cells have a prominent base ment membrane (Fig. 35A) this being reflected in the abundant deposition of basement membrane material in meningiomas. In meningotheliomatous meningiomas, the cell nests are demarcated by a somewhat irregular and occasionally interrupted basement membrane. A striking pattern is noted in fibroblastic meningiomas, where the spindle cells are individually surrounded by irregular and fragmented deposits of basement membrane material (Fig. 35B). In many instances basement membrane components, especially laminin, can be localized in the cytoplasm of neoplastic cells. Furthermore, psammoma bodies appear to contain abundant amounts of type IV collagen and lami nino Although this finding does not rule out a vascular origin for these structures, it is certainly consistent with a meningotheliomatous origin. Given the somewhat irregular pattern of basement membrane deposition in meningiomas, it has appeared impossible to detect characteristic differences in basement membrane patterns between benign and malignant meningiomas.


A

.. , .

.-.

~ Fig. 35. Pattern of basement membrane staining of meninges (A) and of fibroblastic meningeoma (B). In B individual spindle cells are separated by fragments of basement membrane material. Type IV collagen immunohistochemistry (A = 80, B = 250).

:ig.36. Type IV collagen immunoreactivity in a neurofibroma. Note the recticular network of ,asement membranes betweeen the tumor cells around vascular structures (V). - (x 100).


In the peripheral nervous system the tumor type associated with extensive basement membrane deposition is neurofibroma and neurilemmoma (Schwannoma). LEIva et al. (1989) described the specific occurrence in Schwannomas of merosin, a laminin related protein (MCCOMB and BIGNER 1985; FLEISHMAJER et al. 1985). In principle each cell in these tumors is surrounded by a continuous sheath of basement membrane, which results in a pattern of a delicate reticular network in the tumor (Fig. 36). With regard to the basement membrane pattern characteristic differences do not occur between neurofibroma and neurilemmoma. The tendency to deposit basement membrane mate rial is diminished in malignant Schwannomas and it may be altogether absent in poorly differentiated lesions.

5.9 Mesenchymal tissues 5.9.1 Bones and joints

Although bone is the tissue with the most prominent extracellular matrix in the entire body basement membranes do not playa role of any significance here. They only occur in vascular structures and, consequently, basement membranes are found in vasuclar tumors of bone, including hemangioma and aneurysmal bone cyst. Basement membra nes are also found in the rarely occurring lipomas and neurofibromas. SCARPA et al. (1987) reported the production of laminin and type IV collagen in Ewing's sarcomas. In joints basement membranes occur in the support of the synovial lining cells. Basement membrane deposition is a prominent feature of synoviosarcomas and can be of great help in identifying the nature of the neoplasm, especially if the characteristic biphasic pattern is not apparent. In a typical synoviosarcoma, slit-like spaces (corres ponding to synovial spaces) are lined with columnar or cuboidal cells resembling epithelium. These structures are lined with a prominent and often continuous basement membrane (Fig. 37A) (BARSKY et al. 1983; MIETTINEN et al. 1983; BIREMBAUT et al. 1985). These epithelial-like components merge with spindle cell components. In spindle cell areas individual cells tend to be surrounded with fragments of basement membrane material, which results in a fairly diffuse staining pattern (Fig. 37B). This pattern is retained in monophasic synoviosarcomas (containing only the spindle cell compart ment) and can be rather helpful in distinguishing between e.g. fibrosarcoma and sy nOVlOsarcoma. 5.9.2 Soft tissues

In connection with tumor pathology, the term soft tissue is used for a wide variety of tissue types, including fibrous tissue, adipose tissue, muscle, blood and lymphatic ves sels (and often also synovia and peripheral nerves). Tumors in these tissues are classified


~

/_,

;ig.37. Type IV collagen deposition pattern in synoviosarcoma. A: an epithelial-like configura ion (E) is demarcated by a basement membrane. B: in a spindle cell area a diffuse staining of the xtracellular matrix is found. - (x 160).

ig. 38. Vascular network in a hemangioma, outlined by regular type IV collagen immunoreactive asement membranes. - (x 250).


according to the characteristics of the tumor cells (e.g. fibroma, myoma, lipoma for benign and fibrosarcoma, myosarcoma and liposarcoma for malignant tumors). The tendency to deposit a basement membrane is characteristic of some cell types and tends to be retained in neoplastic conditions. Consequently, in benign tumors, such as leiomyoma, hemangioma and lipoma, basement membranes are abundantly present (MIETTINEN et al. 1983; D'ARDENNE et al. 1984; OGAWA et al. 1986). Their pattern of occurrence occasionally helps in recognizing the tissue architecture, for whereas in leiomyomas individual cells are demarcated by a basement membrane, in heman ginomas vascular network is apparent (Fig. 38). Basement membrane material is usually absent in fibromas. In some conditions, such as fibromatoses, the proliferating fibro blastic cell has a phenotype corresponding to that of a myofibroblast, the former comprizing contractile microfilaments and a basal lamina. In these lesions the extracel lular matrix may contain type IV collagen and laminin. In malignant soft tissue tumors basement membrane immunohistochemistry can be used to reveal general tissue architecture (largely through staining of the vascular infra structure) as well as to classify the neoplasm either into the basement membrane nega tive group (including fibrosarcoma and malignant fibrous histiocytoma) or into the basement membrane positive group (which includes angio-, myo- and liposarcomas). Whether or not malignant fibrous histocytomas contain basement membrane specific extracellular matrix components is still a matter of debate. SaINI et al. (1989) reported type IV collagen and laminin immunoreactivity in malignant fibrous histiocytomas, but our own experience as well as that of BIREMBAUT et al. (1985) and OGAWA et al. (1986) indicates that in this category these components do not occur (Fig. 39). Part of the problem here is that malignant fibrous histiocytoma is a poorly defined category lump ing together poorly differentiated neoplasms of various cytological characteristics. In soft tissue sarcomas the general rule is for the degree of differentiation to be reflected in the tendency to deposit basement membrane material (BOSMAN et al. 1989). In well differentiated angiosarcomas, liposarcomas and (rhabdo- or leiomyo-) sarcomas there may be an abundance of basement membrane material (AuTIo-HARMAINEN et al. 1986). But this may be entirely absent in poorly differentiated high grade lesions. Classification of anaplastic sarcomas is therefore not facilitated by basement membrane immunohistochemistry. The pattern of basement membrane staining may help to dis tinguish between specific subcategories of sarcomas. Hemangiosarcomas display a pat tern of vascular structures, whereas (both benign and malignant) hemangiopericytomas show a pattern of basement membranes surrounding individual tumor cells and, in characteristic cases, there is a radial pattern around capillaries. It has been postulated that basement membranes might be absent in lymphangiosarcomas, but in our experi ence basement membranes can be easily detected in these neoplasms. In Kaposi's sarco ma, the vascular spaces are also demarcated by a distinct basement membrane (Fig. 40). Different subcategories are also distinguished in rhabdomyosarcomas. The classical type shows a variable degree of basement membrane deposits surrounding individual


tumor myoblasts. Basement membranes are less distinct in embryonal and pleomorphic types (AuTIo-HARMAINEN et al. 1986). The alveolar configuration in alveolar rhab :lomyosarcoma is demarcated by an irregular basement membrane. A similar pattern is ,een in alveolar soft part sarcoma.

iig. 39. Lack of type IV collagen immunoreactivity in a malignant fibrous histiocytoma. Only the umor vasculature shows basement membrane staining. - (x 100).

ig.40. Type IV collagen immunoreactivity pattern in a case of Kaposi's sarcoma. The vascular nces, which constitue the bulk of the tumor, are outlined by a basement membrane. - (x 250).


6 Concluding remarks In the preceding chapters we have reviewed the current knowledge concerning the structure and composition of the basement membranes, the interaction between neo plastic cells and the extracellular matrix, in particular the basement membrane, as well as the use of immunohistochemical staining of basement membrane antigens in tumor pathology. In this final chapter we will discuss, in more general terms, the practical use of this approach in diagnostic pathology, and what the expectations are for future developments.

6.1 Diagnostic use of basement membrane immunohistochemistry Basement membrane immunohistochemistry can, generally speaking, be applied to delineating tissue architecture, to recognizing early invasive growth, to providing useful indicators on tumor behaviour, and to differentating between different tumor subtypes. 6.1.1 General tissue architecture

One of the most useful aspects of basement membrane immunohistochemistry is that it highlights general tissue architecture. Especially important in this context is that the pattern of vascularization of a neoplasm is clearly outlined. In this fashion, the stromal component and the epithelial compartment of a neoplasms can be clearly distinguished. The delineation of the epithelial compartment on the basis of a basement membrane also facilitates recognition of the general tissue architecture. It is not easy to indicate the exact extent to which this aspect of basement membrane immunohistochemistry con tributes to the diagnostic process. What, however, emerges is that tumor diagnosis is a process of pattern recognition, with tissue architecture figuring as a very important characteristic. Any technique which elucidates tissue architecture will appear as useful. In this regard, basement membrane immunohistochemistry is comparable to the con ventional reticulin staining procedures by means of silver impregnation. 6.1.2 Invasive growth

For histodiagnostic purposes the recognition of invasive growth remains one of the most essential elements in establishing the malignant nature of a neoplasm. Recognizing invasive growth can be significantly facilitated by basement membrane· immunohis tochemistry. Several examples which have already been discussed clearly illustrate this pomt. The differentiation between sclerosing adenosis of the breast and tubular carcinoma can in fact be very difficult. This is probably one of the practical problems in diagnostic


pathology that has most highly benefited from basement membrane immunohis tochemistry. In sclerosing adenosis the epithelial tubules are always surrounded by a continuous and frequently even thickened basement membrane. Even in invasive forms of sclerosing adenosis, e.g in perineural spaces, a discrete continuous basement mem brane is found. In contrast tubular carcinomas of the breast are often completely lacking in basement membranes. Similarly, in chronic pancreatitis distorted ductular structures, which are embedded in an abundant fibrous reaction, may mimic adenocarcinoma, which exhibits a desmoplastic reaction. In this situation, a significant lack of basement membranes surrounding the distorted ductules is indicative of adenocarcinoma. Another problem area which benefits from basement membrane histochemistry is the recgonition of early invasive growth in preneoplastic lesions. Basement membrane staining, which may reveal discrete interruptions of the basement membrane charac teristic for invasive growth, has provided new tools for solving this problem. Typical ~xamples here are: cervical intraepithelial neoplasia and its progression to invasive ;quamous cell carcinoma; non-invasive (To) versus invasive (Tia) bladder carcinoma; ;arcinoma in situ of the larynx and its progression to invasive squamous cell carcinoma; )orderline neoplasms of the ovary and their differentiation from adenocarcinomas (pre fominantly serous); and finally, the progression of atypical endometrial hyperplasia to :ull-fledged endometrial adenocarcinoma. For these latter categories, it has to be taken nto consideration that, although widely recognized as useful, basement membrane listochemistry is not more than a promising new tool and the validity of this approach las to be ascertained by clinicopathological comparison. ;.1.3 Prognosis

One of the important foci of current histopathological research is the establishment )f indicators to reliably predict the most likely course of the disease. Information lerived from these indicators would enable the patient to be more accurately informed 'egarding further expectations for the course of the disease; furthermore it would >ermit formation of subgroups of tumor which might benefit from additional treat nent. In this area several promising reports have been published. Colorectal carcinomas :haracterized by extensive deposition of basement membrane material have been proven o have a somewhat more favorable outcome than those with little or no basement nembrane deposition. In bladder carcinoma similar findings have been reported. The hance of bladder cancer recurrence in non-invasive lesions is higher when basement nembranes are interrupted than in lesions featuring continuous basement membranes. ;inally, the prognosis for squamous cell lung cancer has been reported to be more avorable in cases of extensive basement membrane deposition. It remains to be seen lOW this parameter will affect patient management in these tumor types; nevertheless, ~ese findings merit further exploration of the prognostic use of basement membrane eposition in other tumor types.


6.1.4 Differentiation

Although well differentiated neoplasms in general tend to display more basement membrane deposition than poorly differentiated neoplasms, grading of neoplasms ac cording to the extent of basement membrane deposition is not feasible. Likewise, more or less characteristic patterns of basement membranes have been described for some neoplasms, but they are not sufficiently unique to be reliably used for classification purposes. Only in sarcomas is there some help in classification provided by basement membrane staining.

6.2 Basement membranes and the pathobiology of neoplasms Basement membrane immunohistochemistry has in recent years provided a wealth of new information relevant for our understanding of the development of neoplastic growth. We have come to understand that basement membrane remodeling parallels the development of the invasive phenotype of neoplastic cells, preceding the development of metastasis. It has become clear that tumor cells and host stromal elements both play an important role in this process. Tumor cells deposit basement membrane components in reponse to stimuli from the stroma and stromal cells contribute to this process. Tumor cells elaborate proteases which dissolve the basement membrane as well as other elements of the extracelluar matrix, a process which is essential for tumor cell invasion. Stromal cells may also contribute to this process proteases, receptors for proteases, or protease inhibitors. Growing knowledge of the interactions between tumor cells and the extracellular matrix will lead to an improved understanding of the biology of cancer and this will provide new impulses for the clinical management of cancer. Many examples could be cited to illustrate these expectations. The discovery that growth factors, including TGF-B and b-FGF, accumulate in the basement membrane and may modulate tumor growth; the multidomain structure of involving peptide domains which can augment (but also diminish) the adhesion of cancer cells to a basement membrane; and the discovery of the family of integrin receptors, which play an important role in cell-cell and in cell-matrix interactions - may serve to illustrate this point. Antibodies and nucleic acid probes against these components are already available and will increasingly be used in fundamental and applied studies on the interactions between cancer cells and their surrounding environment. These studies will provide new diagnostic tools and stimulate the exploration of alternative treatment strategies.


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Molecular architecture of basement membranes.

Thin basement membranes in minimally abnormal glomeruli.

Colorectal cancer and basement membranes: clinicopathological correlations.

IgG and IgA antigens in human renal basement membranes.

Building from the Ground up: Basement Membranes in Drosophila Development.

Localization of heparan sulfate proteoglycan in basement membranes.

Laminin forms an independent network in basement membranes.

Engineered Basement Membranes for Regenerating the Corneal Endothelium.

Recent observations on ependyma and subependymal basement membranes.

Development of Animal Models to Study Basement Membranes.

Isolation of glycosaminoglycans (heparan sulfate) from glomerular basement membranes.

Lipids associated with bovine kidney glomerular basement membranes.

Assembly, heterogeneity, and breaching of the basement membranes.

Nature of the glycoprotein components of basement membranes.

Penetration of fimbriate enteric bacteria through basement membranes: a hypothesis.

Studies on the filtration properties of isolated renal basement membranes.

Distribution of annionic sites in glomerular basement membranes: their possible role in filtration and attachment.

The use of polyethyleneimine for demonstration of anionic sites in basement membranes and collagen fibrils.

Basement membranes in fetal, adult normal, hyperplastic and neoplastic human prostate.

Antibodies in guinea-pigs immunized with kidney and lung basement membranes.

c mice.

The presence of anionic sites in basement membranes of cerebral capillaries.

An organizing function of basement membranes in the developing nervous system.

SEM and TEM analyses of isolated human retinal microvessel basement membranes in diabetic retinopathy.