APMIS 98: 875-880, 1990

Laminin in testicular germ cell tumours An immunohistochemical study

GRETE KRAG JACOBSEN

Department of Pathology, Gentofte Hospital, University of Copenhagen, Hellerup, Denmark

Jacobsen, G. K. Laminin in testicular germ cell tumours. An immunohistochemical study. APMIS 98: 875-880, 1990.

Thirty-five testicular germ cell tumours comprising 16 yolk sac tumours, 15 embryonal carcinomas and 13 seminomas were examined for the presence and distribution of laminin using an indirect immunoperoxi- dase technique. In addition, nine normal yolk sacs and 23 carcinomas of the lung were studied. All the yolk sac tumours were positively stained for laminin. Both extra- and intracellular staining were found. Hyaline, eosinophilic material present within the tumours was positively stained, although with varying intensity. In 12 out of 15 embryonal carcinomas, laminin was found as a membrane staining but cytoplasmic staining also occurred. In 10 out of 13 classical seminomas, a membrane staining of many tumour cells was found, while cytoplasmic staining occurred in only a few seminomas. In all but one of the yolk sacs, laminin was present in the membrane beneath both the mesoblastic outer cell layer and the visceral endoderm. Intracellular staining was seen in some of the cells in both cell layers. In nine out of 23 carcinomas of the lung, laminin occurred extra- as well as intracellularly. Thus, this study showed that in normal yolk sacs the presence of laminin was not found to be particularly associated with any of the cell layers. Likewise, demonstration of laminin within yolk sac tumours did not define different patterns or subtypes of the yolk sac tumour. In addition, demonstration of laminin was not found to be useful in differentiating either between yolk sac tumours and embryonal carcinomas or between seminomas and non-seminomatous germ cell tumours. The findings add, however, interesting knowledge to histogenesis and embryogenesis.

Key words: Laminin; germ cell tumours; testicular tumours; human yolk sac.

Grete Krag Jacobsen, Department of Pathology, Gentofte Hospital, University of Copenhagen, DK-2900 Hellerup, Denmark.

Laminin is a well-characterized high molecular weight extracellular non-collagenous glycoprotein constituting a major component of basement membranes in adult tissues (1 7, 22). It is synthe- sized in a variety of cell types that have a basement membrane (6, 17). Laminin was primarily isolated from mouse and rat yolk sac carcinomas contain- ing abundant hyaline material (4, 21, 24). It is well-known that the parietal endoderm of the rodent yolk sac secretes large amounts of laminin (2, 14) in contrast to the visceral endoderm which, like that in the human embryo, produces alpha-fe- toprotein (AFP) among other proteins (7, 8).

Received January 15, 1990. Accepted March 19, 1990.

Laminin has been shown to be the first extracellu- lar matrix protein synthesized in the rodent em- bryo, in which it appears already at the 8-cell stage of development (1 5, 25). In the early stages of development, laminin plays a key role in morpho- genesis and differentiation (9, 13).

Although extensively studied in yolk sacs and yolk sac tumours of mice and rats in the last decade, very few studies have dealt with laminin in human yolk sacs and yolk sac tumours or in germ cell tumours as a whole. Ulbright et al. (23) found that laminin might be a good marker for yolk sac differentiation and especially for yolk sac differen- tiation of parietal type, although parietal yolk sac differentiation in the human yolk sac tumour has not previously been accurately defined.

These authors also found that laminin might be

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helpful in differentiating yolk sac tumours and yolk sac tumour components from embryonal carcinomas and germinomas. The present study was undertaken to examine the distribution of laminin in various types of testicular germ cell tumours and particularly in yolk sac tumour components in order to evaluate the occurrence of laminin for diagnostic purposes. In addition, laminin was studied in a series of normal yolk sacs in order to compare the distribution of laminin in the tumour components that are supposed to be yolk sac imitations with the distribution of lamin- in in the normal analogous tissue. Two major questions were addressed: 1) is laminin useful for differentiating between various patterns of human yolk sac tumour? and 2) is laminin a useful marker in differentiating between yolk sac tumours and other types of germ cell tumours?

MATERIALS AND METHODS

The material comprised nine yolk sacs, 6-12 weeks old, obtained from legal abortions or extra-uterine pregnan- cies. Thirty-five cases of testicular germ cell tumour were selected by virtue of their composition: an almost equal number of yolk sac tumours, 16 cases, and embryonal carcinomas, 15 cases, were selected, comprising pure tumours (3 and 5 cases) as well as components of combined tumours (13 and 10 cases). The yolk sac tumours contained various patterns including the reticu- lar-microcystic, the macrocystic, the solid, the glandular- alveolar, the endodermal sinus, and the papillary pattern. Myxomatous, hepatoid, polyvesicular vitelline, and primitive intestinal patterns were not evaluated as these patterns were either only present in small foci or absent. The embryonal carcinomas showed patterns of solid, papillary, and double-layered type. In addition, 13 classical seminomas were included. For comparison with non-germ cell tumours, the distribution of laminin was also examined in 23 carcinomas of the lung comprising various types except small cell carcinoma.

All tissues were fixed in formalin and routinely pro- cessed in paraffin. One or two blocks from each of the tumours were selected for immunohistochemical exam- ination. An indirect immunoperoxidase technique was used after protease digestion for 5 minutes with 0.1 per cent pronase(type XIV (Sigma 5 147)). Theantibody was a polyconal rabbit anti-laminin raised against rat yolk sac tumour (1,24). The antibody was kindly provided by R . Albrechtsen, who has previously made extensive studies with this antibody and shown that it cross-reacts with human laminin (1). Negative controls were per- formed with substitution of normal rabbit serum for the primary antibody. In the sections, internal positive control was staining of vessel walls and/or basement membranes of the seminiferous tubules.

RESULTS

The results are summarized in Table 1 . All but one yolk sac were positively stained for laminin. The positive staining reaction was not found to be particulary associated with the outer mesoblastic cell layer. A positive membrane staining occurred beneath both the mesoblastic cell layer and the visceral endoderm. In the majority of cases the latter was most heavily stained (Fig. 1). In some cases intracellular positive staining was also found in the cells of both layers. A thick membrane like the Reichert’s membrane in the rodent embryo was not seen.

All yolk sac tumours or yolk sac tumour compo- nents were positively stained for laminin (Fig. 2). The reticular-microcystic pattern, which was the most common pattern, was heavily stained. The positive staining reaction occurred extracellularly with a fibrillar or band-like appearance but intra- cytoplasmic staining was also seen. The solid pattern of yolk sac tumour which usually emerges with the microcystic showed only slight or no positive staining (Fig. 3). The macrocystic pattern was stained very much like the microcystic.

The patterns of glandular-alveolar, endodermal sinus and papillary type contained large amounts of laminin both extra- and intracellularly (Fig. 4 and 5) . The hyaline eosinophilic extracellular material often present in these patterns was posi- tively stained (Fig. 5 ) . Apart from the solid type being negative, the immunohistochemical stain- ings for laminin in the various patterns of yolk sac tumour did not differ essentially, although staining

TABLE 1. Immunohistochemical demonstration of la- minin

No. of Tissues No. of cases positive cases

Yolk sacs 9 8

Yolk sac tumours 16 16

Embryonal carcinomas 15 12

Seminomas 13 10

Carcinomas of the lung 23 9

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intensity varied. Twelve of fifteen embryonal car- cinomas were positively stained for laminin. The positive staining reaction was seen as a fine pericel- lular membrane staining of the tumour cells, but cytoplasmic staining was also present (Fig. 6). The three negative embryonal carcinomas were all of solid type.

In ten of thirteen classical seminomas a delicate pericellular membrane staining of many of the seminoma cells was found (Fig. 7). Only slight cytoplasmic staining occurred in occasional cells in a few seminomas. The laminin-negative semi- nomas did not differ morphologically from those positively stained.

In only nine out of 23 carcinomas of the lung was laminin demonstrated. The positively stained carcinomas included squamous cell carcinomas, adenocarcinomas, and large cell carcinomas. Both extra- and intracellular staining were found. In squamous cell carcinomas, the intracellular stain- ing was most pronounced in the cells at the periphery of the tumour islands (Fig. 8).

It should be mentioned that positive staining of vessel walls was seen in all tumours whether the tumour cells were found to be positive or negative for laminin staining.

DISCUSSION

In the present study of testicular germ cell tu- mours, laminin was demonstrated in all the yolk sac tumours studied, which included the most common patterns. Laminin was found to be present in large amounts in the same patterns that have previously been found to contain AFP (12). Yolk sac patterns with tumour cells surrounded by or containing laminin seemed to be intimately intermingled with, if not the very same as those containing AFP. Likewise, in the solid pattern of yolk sac tumours which contain no AFP or only a small amount, no or only slight positive staining for laminin was found. AFP is known to be synthesized in endodermal cells of the yolk sac (8) as well as in endodermal derivatives in germ cell tumours (12). In the normal yolk sacs studied, laminin was found in association with the visceral endoderm as well as with the mesoblastic outer layer of the yolk sac. An analogue to the Reichter’s membrane present in the rodent yolk sac was not found in the human yolk sacs, and this is in accordance with previous studies of the normal

yolk sac of the human embryo (10, 16). In the rodent yolk sac, Reichter’s membrane is a thick membrane located between the trophoblast and the parietal yolk sac, containing large amounts of laminin (14, 20). In this context it should be stressed that the structure of the human yolk sac differs from that of the rodent yolk sac. Both yolk sacs have a visceral endoderm, but a parietal endoderm only occurs in the rodent yolk sac while the outside of the human yolk sac is covered with mesoblastic cells ( 16). Consequently tumour derivatives of human and rodent yolk sacs would be expected to show different appearances and a parietal yolk sac tumour variant of the human yolk sac tumour is especially not expected. Dalnjanov et al. ( 5 ) have, however, reported a human yolk sac tumour variant with similarities to the rodent parietal yolk sac carcinoma. Although a parietal endoderm does not occur in the normal human yolk sac, imitations of rodent parietal yolk sac within the human yolk sac tumour cannot be fully excluded, but the mere presence of laminin does not prove that it does occur. On the other hand, the well-known endodermal sinus structure of Schiller-Duval body of the human yolk sac tu- mour has been likened to and interpreted as equivalent to the Schiller-Duval body of the nor- mal rat placenta, although such a structure does not occur at any stage during normal human development.

Intracellular demonstration of laminin has been described in rodents in the normal endodermal cells of the parietal yolk sac and in the cells of parietal yolk sac carcinomas in both light micro- scopic and ultrastructural studies (2, 14, 18, 19). In contrast, immunohistochemical demonstration of intracellular laminin has not been reported in studies of human germ cell tumours (3, 5, 23). Ultrastructural evidence of intracellularly located laminin was, however, found by Ulbright et al. (23). The reasons for the lack of reports on intracellularly located laminin may be either the limited number of germ cell tumours investigated or the method of demonstration, which was by an immunofluorescence technique (3, 5) . The fluo- rescence staining signal of extracellularly located laminin may have drowned a less pronounced intracellular staining. Very few studies have dealt with the occurrence of laminin in embryonal carcinomas. Birernbrut et al. (3) found no staining in one embryonal carcinoma and Ulbright et al. (23) found no staining reaction in three cases of

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embryonal carcinomas. Embryonal carcinoma cells show morphological and immunohistochem- ical similarities with the cells of the morula and the blastocyst of very early human development (1 1). Laminin is known to occur as the very first extracellular matrix protein in the mouse embryo, appearing already at the 8-cell stage (15, 25). Demonstration of laminin in the very early stage of development of the human embryo has not been performed, but it is likely that laminin is also present early in the human embryo, and likewise it might be expected to be present in embryonal carcinomas. In the present study, laminin could not be demonstrated in three of the embryonal carcinomas of solid type, which is the less differen- tiated type of the non-seminomatous germ cell tumours. Laminin was, however, present in all the more differentiated patterns of embryonal car- cinoma, mainly extracellularly but in some cells also intracellularly. These findings may reflect the occurrence of laminin in the normal embryogene- sis, indicating that laminin appears early but per- haps somewhat later than in the rodent embryo. This remains, however, to be investigated.

In studies of various other human carcinomas, such as carcinomas of the breast and of the endometrium, laminin has been demonstrated both extra- and intracellularly ( 1, 6). Likewise

staining for laminin in some of the carcinomas of the lung in the present investigation showed both intra- and extracellular staining reaction. In the above-mentioned studies the most poorly differen- tiated carcinomas exhibited cytoplasmic staining although little or no basement membrane staining was found, indicating that the ability to synthesize basement membrane components in these tu- mours is retained (6). These findings show that laminin cannot be used to differentiate between embryonal carcinomas and various other types of carcinomas, some of which may pose differential diagnostic problems, especially in cases of metastatic disease.

The demonstration of laminin in classical semi- nomas has not been published previously. The origin of synthesis of laminin in seminomas is not proven in the present series, although the presence of intracellular staining in occasional cells indi- cates tumour cell origin. The presence of laminin within seminoma cells is under further investiga- tion.

In conclusion, the results of the present study show that the immunohistochemical demonstra- tion of laminin in yolk sac tumours and in embryonal carcinomas is not a helpful tool in differentiating either between the various patterns of yolk sac tumours or between yolk sac tumours

Fig. 1. Normal yolk sac. Positive staining for laminin is seen as a fine membrane staining beneath the endoderm and in the walls of the vessels present between the endoderm and the mesoblastic outer layer. Immunoperoxidase method and haematoxylin. x 250. Fig. 2. Yolk sac tumour with microcystic or reticular pattern. Positive staining for laminin occurs within many tumour cells. Immunoperoxidase method and haematoxylin. x 250. Fig. 3. Yolk sac tumour with solid pattern (left) and microcystic pattern (right). The latter is positively stained for laminin, while the solid pattern is laminin negative. Immunoperoxidase method and haematoxylin. x 250. Fig. 4. Yolk sac tumour with endodermal sinus structure. Positive staining for laminin occurs within the cells covering the perivascular structure and in the cells of the surrounding tumour tissue. Immunoperoxidase method and haematoxylin. x 400. Fig. 5. Yolk sac tumour with papillary pattern. Positive staining for laminin is present within many tumour cells and in the extracellular material. Immunoperoxidase method and haematocylin. x 250. Fig. 6 . Embryonal carcinoma of solid type. Positive staining for laminin is seen as a fine pennuclear membrane especially in the centre of the figure, representing the periphery of the tumour. Immunoperoxidase method and haematoxylin. x 400. Fig. 7. Seminoma, classical type. Positive staining for laminin is demonstrated as delicate membranes between some of the seminoma cells. Heavy staining is also seen in the vessels. Immunoperoxidase method and haematoxylin. x 250. Fig. 8. Squamous cell carcinoma of the lung. Positive staining for laminin is seen in the cytoplasm of tumour cells at the periphery of tumour islands growing in a loose stroma with lymphocytic infiltration. Immunoperoxidase method and haematoxylin. x 250.

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and embryonal carcinomas. Furthermore, laminin is not a helpful marker for differentiating between seminomas and non-seminomatous germ cell tu- mours. Finally, laminin is not a helpful marker for differentiating between germ cell tumours as a whole and various other carcinomas that may give rise to differential diagnostic problems. The find- ings add, however, interesting knowledge to the histogenesis of germ cell tumours and to embryo- genesis.

The author thanks Dr Reidar Albrechtsen, Pathological Anatomical Institute, The Teilum Building, University of Copenhagen, for providing the antibody. IIse Ravn is acknowledged for typing the manuscript and Dr Hans Lyon for correcting the English language.

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