Basement membrane structures in tumors of the ovary

357

Europ. J. Obstet. Gynec. reprod. Biol., 20 (1985) 357-371 Elsevier

EIO 00256

Basement membrane structures in tumors of the ovary Frej Stenb;ick ’ and Veli-Matti Wasenius * ’ Department of Pathology, Universityof Oh, Nordic Councilfor Arctic Medical Research, Oulu, and 2 Radiotherapy

Clinic and Oncology, University of Helsinki, Helsinki, Finland Accepted

for publication

20 May 1985

STENBACK, F. and WASENIUS, V.-M. (1985): Basement membrane structures in tumors of the ovary. Europ. J. Obstet. Gynec. reprod. Biol., 20, 357-371. The location and amount of the basement membrane (BM) components collagen IV and laminin were studied in ovarian epithelial, sex cord-stromal and germ cell tumors. BM structures were found in the epithelial stromal interface of benign surface epithelial tumors and, though discontinuous, around well-differentiated tumor islets, being less well developed in invasive undifferentiated neoplasms. The stromal components in Mtillerian mixed tumors had less distinct BM structures, a finding useful for the classification of these neoplasms. Thecomas and fibromas had scanty collagen IV and laminin; granulosa cell tumors contained large amounts of BM material. A fine diffuse BM-positive pattern occurred in dysgerminomas and endodermal sinus tumors; BM structures in cystic teratomas were distinct. Collagen IV and laminin were well-developed in benign and slow-growing tumors with epithelial components and in their metastases, but less distinct in stromal tumors and highly malignant undifferentiated tumors, showing the usefulness of this method for the clinical and biological classification of such tumors.

basement

membrane;

collagen

IV; tumors

Introduction

The accurate diagnosis and classification of ovarian tumors can be difficult when based on light microscopy, and is subject to various interpretations (Blaustein, 1977). Immunohistochemical markers have recently been suggested as diagnostic aids to cellular differentiations (Km-man et al., 1984; Miettinen et al., 1983). These include the demonstration of keratins for epithelial cells (Nagle et al., 1983), myoglobin for skeletal muscle differentiation (Mukai et al., 1980), Factor VIII-related antigen for endothelial cell differentiation (Mukai et al., 1980), alpha-fetoprotein (Palmer et al.,

Address correspondence and reprint requests to: Dr. Frej Stenback, 90220 Oulu, Finland; Tel. 358(81)334202.

OOZS-2243/85/$03.30

0 1985 Elsevier Science Publishers

University

B.V. (Biomedical

of Oulu, Kajaanintie

Division)

52 D,

358

1976; Wilkinson et al., 1973), carcinoembryonic antigen (Marchand et al., 1975; Fenoglio et al., 1981), and human chorionic-gonadotropin (Kurman and Scardino, 1981) for germ cell tumors and steroids for hormonally active tumors (Kurman et al., 1979) as well as intermediate filaments such as vimentin (Miettinen et al., 1983) for stromal tumors. Connective-tissue cells also produce an extracellular matrix composed of various collagenous proteins and non-collagenous substances (Martinez-Hernandez and Amenta, 1983). Basement membrane (BM) -type proteins have been described in soft-tissue tumors (Miettinen et al., 1983) and epithelial tumors, notably those of the mammary gland, where they have been associated with tumor behaviour and classification (Liotta et al., 1979; Albrechtsen et al., 1981). BM penetration and disruption have been considered crucial for tumor advance and malignant behaviour in the case of epithelial tumors (Liotta et al., 1980, 1983). The main intrinsic BM components synthesized and secreted by cells resting on the BM include type IV collagen, laminin, heparan sulphate, proteoglycan and possibly entactin (MartinezHernandez and Amenta, 1983). Type IV collagen is composed of 3 chains interrupted by segments with sequences other than glycine, and large amounts of 3-hydroxyproline carbohydrate sidechains (Timpl et al., 1981). Type IV collagen contains a unique region, the 7s domain, which is relatively resistant to bacterial collagenase, and is the site pf crosslinking among type IV collagen molecules (Risteli et al., 1980). Laminin, is a, non-collagenous component of BM (Risteli and Timpl, 1981), a glycoprotein with a molecular weight of 900000 which has a cross-like shape, having one long and three short arms, and a non-distinctive amino-acid composition (Timpl et al;, 1979). Antibodies to laminin and type IV protein are both stained exclusively in the BM (Martinez-Hemandez and Amenta, 1983) when using immunoelectronmicroscopical techniques. The present work concerns the expression of BM proteins in ovarian tumors using antibodies against type IV collagen and laminin. The purpose was to elucidate the role of these structures in the histogenesis and development of different types of ovarian tumors and to determine their usefulness for clinical diagnosis and classification.

Material and methods

Tissue samples

Altogether 101 specimens obtained at surgery from 97 patients with ovarian tumors were studied. The specimens were fixed in 10% buffered formalin, embedded in ,paraffin and routinely stained with hematoxylin and eosin. The lesions were classified using the standard histopathologic criteria (Blaustein, 1977). Periodic acid-Schiff staining (PAS) was also used in some cases for visualization of the basement membrane, as well as other stains such as Masson’s and van Gieson’s stain when needed. The clinical behaviour of the lesions, including their gross appearance, symptoms and progress, was known from follow-up.

359

Antibodies

The 7s collagen domain of human type IV collagen was purified from human kidney (Risteli et al., 1980) and the fragment Pl of laminin from human placenta (Risteli and Timpl, 1981) as described previously. Antisera to these proteins were raised in rabbits. The antibodies were purified by immunoabsorption with the relevant antigen coupled to Sepharose 4B. The laminin antibodies were crossabsorbed with 7s collagen and vice versa, and there was no cross-reaction between the two antibodies in the radioimmunoassay. Immunoperoxidase

staining

For the immunohistochemical studies the avidin-biotin modification (Hsu et al., 1981) of the peroxidase-antiperoxidase method was used. 5-pm-thick paraffin sections were deparaffinized and treated with 0.4% pepsin (Merck, Darmsiadt, F.R.G). Endogenous peroxidases were blocked by incubating with H202. The sections were successively treated with rabbit antibodies, biotinylated anti-rabbit immunoglobulin (Vector Laboratories, Burlingame, CA), dilution 1:500, avidin (Vector), dilution 1: 1000, and biotinylated horseradish peroxidase complex (Vector). The peroxidase reaction was performed using 3-amino-9-ethylcarbazole (Sigma Chemical Co., St. Louis, MO) as a substrate.

Fig. 1. Well-differentiated mutinous cystadenocarcinoma with distinct laminin-positive branes stained with antibodies to fragment Pl of human laminin; X 180.

basement

mem-

Fig. 2. Mutinous adenocarcinoma showing collagen IV ( + ) -PC tsitive basement membranes, lacking around invading tumor cell, stained with antibodies to the 7S domalin of human type IV collagen; x: 180.

Rt!SllltS In benign surface epithelial tumors such as mutinous and serous cystadenomas and in endometrioid tumors the antibodies stained a narrow band around epithelial structures. This was also seen in malignant epithelial tumors with a high degree of differentiation which displayed a distinct BM in the epithelial/stromal junction positive for collagen IV and laminin (Fig. 1). Disruptions were observed, with lack of staining around invading epithelial islets of malignant cystadenocarcinomas (Fig. 2). Glandular epithelial structures in ovarian adenocarcinomas contained considerable BM material, as shown here (Fig. 3). Lymph node metastases also displayed lamininand collagen IV-positive BM structures around epithelial cells. The distribution and amount of BM structures became less distinct with decreasing differentiation, undifferentiated tumors containing only scanty BM material (Fig. 4).

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Fig. 3. Ovarian adenocarcinoma with epithelial structures and partly preserved stained with antibodies to the 7S domain of human type IV collagen; x 180.

Fig. 4. Ovarian adenocarcinoma ( + ), distinct only in arterial collagen; X 120.

grade III with spotty irregular walls, stained with antibodies

patches of collagen to the 7s domain

basement

membranes

IV-positive of human

material type IV

362

Fig. 5. Epithelial/stromal junction and vessel walls containing collagen-IV positive material in ovarian carcinosarcoma stained with antibodies to the 7s domain of human type IV collagen; x 360.

Fig. 6. Abundant collagen IV-positive material ( + ) in vessel wall of ovarian carcinosarcoma, material in stroma, stained with antibodies to the 7s domain of human type IV collagen; X 360.

scanty

363 TABLE

I

Number

of specimens

Intensity

of staining:

and intensity

of staining

of collagen

I= focal, 2 = discontinuous,

Type of tumor

Number

Mutinous cystadenoma Mutinous cystadenccarcinoma Serous cystadenoma Serous cystadenocarcinoma Endometrioid adenocarcinoma Carcinosarcoma Mixed mesodermal tumor Granulosa cell tumor Thecoma Fibroma Sertoli-Leydig cell tumor Dysgerminoma Embryonal cell carcinoma Endodermal sinus tumor Cystic teratoma Immature teratoma Teratocarcinoma, squamous Metastases from breast, colon and stomach Krukenberg’s tumor

12 6 10 5 5 4 4 8 6 5 2 4 3 3 10 2 2

IV and Iaminin

3 = distinct, of specimens

5 5

in ovarian

tumor

continuous. Intensity

of staining

collagen

IV

laminin

3

3 l-3

l-3 3

3 1 1 2 2 2 2 3 1-2 1

3 1-3 l-3 1 l-2 3 1 1 2 2 2 2 3 l-2 1

l-3 l-3

l-3 1-3

l-3 l-3 1 l-2

101

Fig. 7. Androblastoma stained with antibodies

with Leydig cell component to the 7s domain of human

containing irregular basement type IV collagen; ( + )X 180.

membrane

structures

364

Fig. 8. Abundant collagen IV-positive material in ovarian granulosa cell tumor stained with antibodies to the 7S domain of human type IV collagen; x 180.

Fig. 9. Fine fibrillary basement membrane structures in ovarian dysgerminoma stained with antibodk i to the 7s domain of human type IV collagen; X 180.

365

The ovarian carcinosarcomas consisted of an admixture of malignant epithelial and stromal elements. The epithelial component was glandular in nature, with a distinct collagen IV-positive BM (Fig. 5), while the stroma was made up of tightly packed spindle cells with scanty collagen IV- or laminin-positive material (Fig. 6). Occasionally the glands were anaplastic, bizarre and pleomorphic, with irregular BM structures surrounding the epithelial structures and also as irregular strands within the tumor tissue. The blood vessels were distinctly positive for laminin and collagen IV in the vessel wall (Fig. 6). Sex-cord stromal tumors displayed a varying distribution of BM-like material. Thecomas consisted of intersecting bundles of spindle cells producing collagen, the cells resembling those of nodular stromal hyperplasia. Hyalinized strands of collagen were rarely prominent; reticulin fibres surrounded individual cells, as in normal stroma; BM-like material was scanty. Fibromas, morphologically similar though lipid-negative, also failed to stain for collagen IV or laminin except around blood vessels. The androblastomas, thoug.5 few, contained a distinct tubular component with well-developed Sertoli cells having a distinct collagen IV-positive BM. The Leydig cell component was less developed with irregular BM structures (Fig. 7). The granulosa cell tumors were mostly of the microfollicular type with inconspicuous tumor cells. They lacked blood vessels and relied on vessels running within the network of non-specific stroma. The reticulin fibres surrounded clusters of cells rather than individual cells. The sarcomatoid-type tumors with trabecular areas contained abundant collagen IV-positive material (Fig. 8). Less differentiated tumors also contained collagen IV in variable amounts.

Fig. 10. Strands of collagen IV-positive material of varying thickness in ovarian embryonal cell carcinoma stained with antibodies to the 7s domain of human type IV collagen; X 180.

Fig. 11. Cystic teratoma of the ovary with basement membrane structures in the epithelial/stromal junction of squamous and columnar epithehum, hair follicles, sebaceous glands and vessel walls stained with antibodies to the 7s domain of human type IV collagen; ~45.

Fig. 12. Ovarian squamous-type teratocarcinoma epithelial islets. PASM stain, x 360.

with partly positive basement-like structures around

The germ cell tumors also displayed BM-like material though depending on tumor type. The dysgerminomas consisted of typical oval or round cells with abundant cytoplasm in a distinct stroma, varying from a fine, delicate network to large, fibrous strands. A diffuse, fine reticular staining pattern was seen for both laminin and collagen IV (Fig. 9). In the endodermal sinus tumors the distribution of BM components varied from prominent to unremarkable depending upon the basic architecture. The embryonal carcinomas consisted of solid aggregates of epithelial-like polygonal or ovoid cells in pseudoglandular or cleft-like arrangements. BM-like structures were observed as strands surrounding these structures (Fig. 10). The mesenchymal component varied in composition, being either loose with little collagen IV or laminin-positive material, or cellular with more fibrillar material. The mature cystic teratomas consisted of typical organoid structures with a squamous or columnar epithelium resting on a distinct BM. Laminin and collagen IV were also abundant in the vessel walls and around the hair follicles (Fig. 11) in the interphase of epithelial and stromal tissue components. Collagen IV and laminin were less conspicuous in the immature solid teratomas, and partly present in teratocarcinomas stained by conventional techniques such as PASM (Fig. 12). Basement membrane structures were also found around some epithelial metastases in other organs as previously mentioned. Also primary tumors in distant sites such as rectum, breast or stomach metastasizing to the ovary showed some tumor islets surrounded by basement membrane components laminin and collagen IV. In Krukenberg’s tumor (Fig. 13) irregular strands of collagen IV-positive material were also found in the stroma.

Fig. 13. Krukenberg tumor of the ovary with irregular strands of basement membranes epithelial cells stained with antibodies to the 7S domain of human type IV collagen:

x

surrounding 180.

some

368

Discussion

As demonstrated in this study BM components laminin and collagen IV were easily discernible in ovarian tumors of many different types. They were found in the epithelial-stromal interface, in the stroma of sex-cord stromal tumors such as granulosa cell tumors and in germ cell tumors, i.e. dysgerminomas and teratomas. They were produced by normal, metaplastic, dysplastic and teratomatous cells to varying degrees; the distribution and intensity were, however, dependent on the tumor type. The most variable distribution of BM material was found in epithelial neoplasms, with a distinct layer of collagen IV- and laminin-positive material in benign cystadenomas, whether mutinous or serous in origin, similar to benign tumors of the mammary gland (Liotta et al., 1979; Albrechtsen et al., 1981) and skin (Kallioinen et al., 1984). The malignant epithelial tumors, depending upon the degree of differentiation, also displayed a decreasing amount of BM-positive structures, seen also in EM studies of the ovary (Stenback, 1981;) and in tumors of other organs by immunohistochemistry (Martinez-Hernandez and Amenta, 1983; Liotta et al., 1979; Albrechtsen et al., 1981; Stenback et al., 1985). Epithelial structures in mixed tumors were also distinctly visualized by collagen IV and laminin antisera, while the sarcomatous components were mostly negative. Mixed tumors included carcinomas with sarcomatous areas, carcinomas with a benign reactive mesenchymal component (pseudosarcoma) and combined carcinomatous and sarcomatous lesions; a heterogenous group of neoplasms with varying clinical characteristics and prognosis (Barwick and LiVolsi, 1979; Dehner et al., 1971). BM analysis was useful in separating the epithelial and stromal component. In addition, heterologous structures, known to be associated with a worsening prognosis (Blaustein, 1977) were easily seen surrounded by BM structures. Sex-cord stromal tumors in this study contained scanty BM-positive material. In the Miettinen et al. (1983) study of soft-tissue tumors such as leiomyomas, leiomyosarcomas, schwannomas and neurofibromas were laminin-positive, while dermatofibromas and malignant fibrous histiocytomas were negative. Mesenchymal tumors in this study were essentially negative whether fibromas or thecomas. Laminin and collagen IV analysis in this study did not, however, separate different stromal tumors. Other tumors of the sex-cord stromal tumor group in this study such as granulosa cell tumors were strongly positive when stained for collagen IV and laminin. This may ba related to the histological structure as they were mostly of the sarcomatoid type. Whether this finding has a prognostical significance remains to be determined. It would, however, be of potential use since, at present, reliable prognostical morphological criteria are not generally agreed upon (Young and Scully, 1982). Sertoli-Leydig cell tumors also contained distinct BM-positive areas; however, the number was small and the tumors were of the well-differentiated forms. Differentiation was related to survival in most studies (Novak and Long, 1965; Roth et al., 1981) and it may be concluded, in congruence with epithelial tumors, that the occurrence of BM structures indicates a favourable prognosis. Ultrastructural studies have also shown BM structures in this type of tumor (Tavassoli et al., 1980).

369

BM structures were also discernible in germ cell tumors regardless of type. Dysgerminomas, endodermal sinus tumors and embryonal carcinomas have a high degree of malignancy (Kurman and Norris, 1977). As shown here they were BM-positive, indicating that BM structures per se are not indicators of favourable prognosis as in epithelial tumors of the ovary. Tumor markers such as carcinoembryonic antigen, alpha-fetoprotein and chorionic gonadotropin have been useful in the diagnosis and clinical follow-up of ovarian tumors (Palmer et al., 1976; Wilkinson et al., 1973; Marchand et al., 1975; Fenoglio et al., 1981; Kurman and Scardino, 1981; Talerman and Haije, 1974). BM analysis may be used to separate pure dysgerminomas from tumors containing other germ cell components. as pure tumors have a much better prognosis (Asadourian and Taylor, 1969). As shown in this study, BM production was also preserved in teratomas in the interface of epithelial and stromal structures. Regardless of epithelial cell type, squamous, columnar, transitional or other, laminin and collagen IV production was preserved. Thus BM production was a property of the cell depending only on type. not on location or development. Immature, malignant teratomas are often ill-defined as they are composed of immature tissues differentiated to one or all of the three germ layers. New classifications (Nogales et al., 1976) allow for more precise definition, and they may be aided by BM studies as the amount of collagen IV and laminin was related to the degree of maturation of the tumor. As shown in this study, BM analysis may be of value in the classification of poorly differentiated neoplasms, which is difficult when based on histology alone. Identification of the cell of origin of the neoplasm was also aided by BM analysis for epithelial neoplasms as well as identification of different stromal components. BM analysis for clinical purposes as an indicator of malignancy and as a tool for classification of tumors was useful in several situations, as shown in this study. More studies are, however, needed before final conclusions can be made. Technical specifications have also to be established, as fixation, specimen preparation and antisera affect the results and their interpretation (Martinez-Hernandez and Amenta, 1983; Liotta et al., 1983). The antisera in this study were prepared against human collagen IV and laminin; most published studies to far have used murine antigens (Miettinen et al., 1983; Liotta et al., 1979; Albrechtsen et al., 1981; Liotta et al., 1980; Liotta et al., 1983; Barsky et al., 1983). Previous studies using conventional stains such as PAS or reticulin have yielded some information on the occurrence of BMs in ovarian tumors (Blaustein, 1977). These stains, however, delineate various components which are not BMs; the PAS stain shows mainly the lamina rara, while reticulin stains interstitial-type collagens, namely collagen type I and type III (Stenback et al., 1985). As shown also in this study these stains, as well as modifications such as PASM, stain basement-like structures only to a limited extent.

Acknowledgements

The authors gratefully acknowledge the gift of collagen IV and laminin antisera from Dr. Juha Risteli and Dr. Leila Risteli of the Collagen Research Unit, Departments of Clinical Chemistry and Medical Biochemistry, University of Oulu, Finland.

370

References Albrechtsen, R., Nielsen, M., Wewer, U.. Engvall, E. and Ruoslahti, E. (1981): Basement membrane changes in breast cancer detected by immunohistochemical staining for laminin. Cancer Res., 41, 5076-5081. Asadourian, L.A. and Taylor, H.B. (1969): Dysgerminoma. An analysis of 105 cases. Obstet. and Gynec., 33, 370-379. Barsky, S.H., Siegal, G.P., Janotta, F. and Liotta, L.A. (1983): Loss of basement membrane components by invasive tumours but not by their benign counterparts. Lab. Invest., 49, 140-147. Barwick, K.W. and LiVolsi, V.A. (1979): Malignant mixed Miillerian tumours of the uterus: A clinicopathologic assessment of 34 cases. Amer. J. surg. Path., 3, 125-135. Blaustein, A. (1977): Pathology of the Female Genital Tract. Springer, New York. Dehner, L.P., Norris, H.J. and Taylor, H.B. (1971): Carcinosarcomas and mixed mesodermal tumours of the ovary. Cancer, 27, 207-216. Fenoglio, C.M., Crum, C.P., Pascal, R.R. and Richart, R.M. (1981): Carcinoembryonic antigen in gynecologic patients. II. Immunohistological expression. Diagnostic Gynec. Obstet., 4, 291-9. Hsu, S., Raine,,L. and Fanger, H. (1981): A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Amer. J. clin. Path., 75, 734-738. Kallioinen, M., Autio-Harmainen, H., Dammert, K., Risteli, J. and Risteli, L. (1984): Discontuinity of the basement membrane in fibrosing basocellular carcinomas and basosquamous carcinomas of the skin: an immunohistochemical study with human laminin and type IV collagen antibodies. J. invest. Dermatol., 82, 248-251. Kurman, R.J., Ganjei, P. and Nadji, M. (1984): Contributions of immunocytochemistry to the diagnosis and study of ovarian neoplasms. Review. Int. J. Gynec. Path., 3, 3-26. Kurman, R.J., Goebelsman, U. and Taylor, C.R. (1979): Steroid localization in granulosa-theta tumours of the ovary. Cancer, 43, 2377-84. Kurman, R.J. and Norris, H.J. (1977): Malignant germ celltumours of the ovary. Human Path., 8, 551-564. Kurman, R.J. and Scardino, P.T. (1981): Alpha-fetoprotein and human chorionic gonadotropin in ovarian and testicular germ cell tumours. In: Diagnostic Immunocytochemistry, pp. 277-98. Editor: R.A. De Lellis. Masson Publishing USA, New York. Liotta, L.A., Foidart, J.M., Robey, P.G., Martin, G.R. and Gullino, P.M. (1979): Identification of micrometastasis of breast carcinomas by presence of basement membrane collagen. Lancet, ii, 146-147. Liotta, L.A., Rao, C.N. and Barsky, S.H. (1983): Tumor invasion and the extracellular matrix. Lab. Invest., 49, 636-649. Liotta, L.A., Tryggvason, K., Garbisa, S., Hart, I., Foltz, C.M. and Shafie, S. (1980): Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature, 284, 67-68. Marchand, A.. Fenoglio, CM., Pascal, R., Richart, R.M., and Bennett, S. (1975): Carcinoembryonic antigen in human ovarian neoplasms. Cancer. Res., 35, 3807-10. Martinez-Hernandez, A. and Amenta, P.S. (1983): The basement membrane in pathology. Lab. Invest., 48, 656-677. Miettinen, M., Foidart, J.M. and Ekblom, P. (1983): Immunohistochemical demonstration of laminin, the major glycoprotein of basement membranes as an aid in the diagnosis of soft tissue tumors. Amer. J. Clin. Path., 79, 306-311. Miettinen, M., Lehto. V.-P. and Virtanen, I. (1983): Expression of intermediate filaments in normal ovaries and ovarian epithelial, sex-cord stromal, and germinal tumours. Int. J. Gynec. Path., 2, 64-71. Mukai, K., Rosai, J. and Burgdorf, W.H.C. (1980) Localization of factor VIII-related antigen in vascular endothelial cells using an immunoperoxidase method. Amer. J. surg. Path., 4, 273-276. Mukai, K., Varela-Duran, J. and Nochomovitz, L.E. (1980): The rhabdomyoblast in mixed mulletian tumours of the uterus and ovary: an immunohistochemical study of myoglobin in 25 cases. Amer. J. clin. Path., 74, 101-4. Nagle, R.B., Clark, V.A., McDaniel, K.M. and Davis, J.R. (1983): Immunohistochemical demonstration of keratins in human ovarian heoplasms. A comparison of methods. J. Histochem. Cytochem., 31, 1010-4.

371 Nogales, F.F., Favara, B.E., Major, F.J. and Silverberg, S.G. (1976): Immature teratoma of the ovary with a neural component (‘solid’ teratoma). A clinicopathologic study of 20 cases. Human Path.. 7. 625-642. Novak, E.R. and Long, J.H. (1965): Arrhenoblastoma of the ovary. A review of the ovarian tumor registry. Amer. J. Obstet. Gynec., 92, 1082-93. Palmer, P.E., Safaii, H. and Wolfe, H.J. (1976): Alpha-1-anti-trypsin and alpha-fetoprotein: protein markers in endodermal sinus (yolk-sac) tumours. Amer. J. Clin. Path., 65, 575-582. Risteli, J., Bachinger, H.P., Engel, J., Furthmayr, H. and Timpl, R. (1980): Characterization of an unusual basement membrane structure. Europ. J. B&hem., 108, 239-250. Risteli, L. and Timpl, R. (1981): Isolation and characterization of pepsin fragments of laminin from human placental and renal basement membranes. Biochemistry, 193, 749-755. Roth, L.M., Anderson, MC., Govan, A.D.T., Langley, F.A., Gowing, N.F.C. and Woodcock, A.S. (1981): Sertoli-Leydig cell tumours. A clinicopathologic study of 34 cases. Cancer, 48, 187-97. Stenback, F. (1981): Benign, borderline and malignant serous cystadenomas of the ovary. A transmission and scanning electron microscopical study. Pathology: research and practice, 172: 58-72. Stenback, F.. Wasenius, V.-M., Risteli, J. and Risteli, L. (1985): Basement membranes in progressing intraepithelial cervical neoplasia, an ultrastructural and immunohistochemical study with antibodies against human type IV collagen and laminin. Gynec. Obstet. Invest., in press. Talerman, A. and Haije, W.G. (1974): Alpha-fetoprotein and germ cell tumours: A possible role of yolk sac tumour in production of alpha-fetoprotein. Cancer, 34, 1722-1725. Tavassoli, F.A. and Norris, H.J. (1980): Sertoli tumours of the ovary. A clinicopathologic study of 28 cases with ultrastructural observations. Cancer, 46, 2281-97. Timpl, R., Rohde, H., Gehron Robey, P., Rennard, S.I., Foidart. J.M. and Martin, G.R. (1979): Laminin - a glycoprotein from basement membranes. J. biol. Chem., 254, 9933-9937. Timpl, R., Wiedemann, H., Van Delden, V., Furthmayr, H. and Kuhn, K. (1981): A network mode1 for the organization of type IV collagen molecules in basement membranes. Europ. J. Biochem., 120, 203-211. Wilkinson, E.J., Friedrich, E.G. and Hosty, T.A. (1973): Alphafetoprotein and endodermal sinus tumour of ovary. Amer. J. Obstet. Gynec., 116, 711-714. Young, R.H. and Scully, R.E. (1982): Ovarian sex cord-stromal tumours: recent progress. Int. J. Gynec. Pathol., 1, 101-123.