Heterogeneity of intermediate filament expression in human testicular seminomas

Differentiation (1990) 45 :242-249

Differential ion Ontogeny and Neoplasia 0 Springer-Verlag 1990

Heterogeneity of intermediate filament expression in human testicular seminomas Mina Fogel' *, Beatriz Lifschitz-Mercer', Roland Moll 2 , Ilana Kushnir', Nurith Jacob', Rudiger Waldherr 3, Alexander Livoff 4, Werner W. Franke 5, Bernard Czernobilsky

'

' Department of Pathology, Kaplan Hospital, Rehovot 76100, Israel

' Department of Pathology, University of Mainz. Federal Republic of Germany Department of Pathology, University of Heidelberg. Federal Republic of Germany Department of Pathology, Medical School of the Hebrew University and Hadassah, Jerusalem, Israel Division of Membrane Biology and Biochemistry. Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg, Federal Republic of Germany Accepted in revised form October 27. 1990

Abstract. Testicular seminoma has in the past been considered to represent a germ cell tumor incapable of further differentiation. In recent years this view has been challenged on the basis of morphologic and chromosomal studies. Moreover, studies of intermediate filaments (IF) of seminoma cells have provided evidence of the capability of seminoma cells to differentiate in different directions. In the present study of the IF protein profile of 26 human testicular seminomas, using frozen as well as formalin-fixed, paraffin-embedded tissues, we report evidence of a heterogeneous differentiation potential inherent in these neoplasms. Thus, in 4 of the seminomas neither cytokeratins nor vimentin were detected ; 3 showed vimentin positive cells but no cytokeratins; in 4 seminomas only cytokeratins were detected. In the remaining 15 cases both cytokeratins and vimentin were present, with occasional cells demonstrating coexpression of cytokeratin and vimentin. While the cytokeratins present were mostly of the "simple epithelial type", in 2 instances seminoma cells also contained cytokeratins 4 and 17, normally found in stratified and/or complex glandular epithelia. Furthermore, in 3 cases scattered tumor cells stained for desmin and in 2 other seminomas neurofilaments were identified. All of the cases showed variable positive staining for desmoplakins and desmoglein, indicative of the presence of desmosomes. It can therefore be concluded that, while some seminomas seem to be devoid of IFs, most of them show varied differentiation patterns usually with epithelial features but occasionally also with components commonly regarded as characteristic of myogenic or neurogenic differentiation. These observations may help to elucidate the relationship of seminomas to other germ cell tumors, and also contribute to our understanding of the histogenesis of these neoplasms.

*

To whom ofJprint requests should he sent

Introduction

Seminoma is the most common testicular neoplasm, representing in its pure form approximately 40% of all the tumors of the testis [40]. While the germ cell origin of seminoma, like that of other germ cell tumors, is now generally accepted [29], the relationship and potential of differentiation of the various forms of neoplasms within this group of tumors is still being debated. According to Teilum [42] seminoma is a germ cell tumor which has not acquired the potential for further differentiation. This view has, however, been challenged, initially because of the common intimate admixture of seminoma and other, nonseminomatous germ cell tumors. Furthermore, in about 40% of cases, the metastases from apparently pure seminoma contain other than seminomatous germ cell elements [4]. A recent argument in favour of the capacity of seminoma to differentiate into nonseminomatous germ cell tumors has been provided by chromosomal studies [31]. Thus a revision of the histogenesis and interrelationship of germ cell tumors, as was attempted by Skakkebaek et al. [38], appears warranted. During recent years the IF protein profile has emerged as a useful means to study the differentiation patterns of normal and neoplastic cells as well as of tissues [lo]. Seminomas have also been studied with these methods, albeit with a restricted number of antibodies to cytoskeletal proteins. The results showed that these tumors were either completely devoid of cytokeratins [I], or contained predominantly vimentin, with only occasionally cytokeratin-positive cells [9, 24, 341. Nakagawa et al. [30] did not demonstrate cytokeratins or vimentin in gonadal seminomas; however, these IF proteins were present in some instances when the neoplasms were located intracranially. No other kind of I F protein was identified in these studies. In the present study of the IF protein complement

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of testicular seminomas, a large battery of monoclonal antibodies to different IF proteins was employed, using whenever possible in parallel immunofluorescence microscopy on frozen tissues, and immunoperoxide methods on both frozen and formalin-fixed, paraffin-embedded material. This approach has allowed the demonstration of various cytokeratins, of vimentin and also of occasional desmin and neurofilament protein in tumor cells, providing evidence of the varied differentiation potential inherent in these neoplasms.

Table 1. Monoclonal antibodies used on frozen and formalin-fixed, paraffin-embedded tissues

Antibody

Antigens recognized

References and source

LU-s *

Broad range of type 1 and type 11 cytokeratins

[ 131 Boehringer, Mannheim, FRG

KG8.13

Broad range of type 11 Cytokeratins and Cytokeratin 18

[14] BioMakor,

6B10

Cytokeratin 4

[ 151 Eurodiagnostics, Apeldoorn, The Netherlands

CK7

Cytokeratin 7

[ 161 Boehringer, Mannheim, FRG

Ks8.17.2

Cytokeratin 8

[17] Progen Biotechnics, Heidelberg, FRG

K8.1.42

Cytokeratin 8

[18] Progen Biotechnics

K&60*

Cytokeratin 10, 11

[19] BioMakor

iC7

Cytokeratin 13

[I 51 Eurodiagnostics, Apeldoorn The Netherlands

Ks13.1*

Cytokeratin 13 (14, 16)

[20] Progen

E3

Cytokeratin 17

[21] Kindly provided by Dr. S.M. Troyanovsk y

Ks18.174

Cytokeratin 18

[21] Progen

CK-5 *

Cytokeratin 18

Methods Tissues. Samples from 26 surgically removed seminomas were fixed in 10% buffered formalin and embedded in paraffin. In 18 of the cases tissue samples were also snap-frozen in isopentane, which was precooled in liquid nitrogen and stored at -70" C. Diagnoses were reached from hematoxylin-eosin (HE)-stained slides of the paraffin-embedded tissues. Antibodies. Primary monoclonal antibodies used are listed in Table 1. In one case in which tumor cells stained positively with the desmin antibody from BioMakor [7], three additional monoclonal desmin antibodies were used : DE-CJ-I0 (Camon; Wiesbaden, Federal Republic of Germany, FRG), DE-B-5 (Boehringer, Mannheim, FRG) [7] and 033 (Dakopatts, Hamburg, FRG) [46]. In this case the tumor was also stained using monoclonal skeletal myosin antibody MY32 [18] and monoclonal tropomyosin antibody TM311 (both from BioMakor). The secondary antibodies used for immunofluorescence were fluorescein-isothiocyanate (F1TC)-coupled or Texas-red-coupled goat antibodies to immunoglobulins of guinea pig or mouse (Dianova, Hamburg, FRG). Secondary antibodies for immunoperoxidase staining were biotinylated antibodies to mouse immunoglobulins and avidin-peroxidase complex supplied as a kit (Vectastain, Elite, ABC Kit; Vector Lab, Burlingame, Ca, USA). Immunofluorescence.Cryostat sections 4-5 pm thick were collected on glass slides, air-dried, fixed in acetone at -20" C for 10 min, and exposed to primary and secondary antibodies as previously described [ 1 21. Antibody-stained sections were dehydrated in absolute ethanol, mounted with Entellan (Merck, Darmstadt, FRG) and examined with a Zeiss Axiophot microscope equipped for epifluorescence with a planapochromat x 40/1.0 iris objective. For double-label-cytokeratin-vimentin immunofluorescence microscopy, guinea-pig cytokeratin antibodies G P 11 against cytokeratins 8 and 18 (Progen, Heidelberg, FRG) were used, together with the monoclonal murine vimentin antibody (Table 1). Both primary antibodies were applied simultaneously, as were the secondary antibodies [28].

Rehovot, Israel

C Y-90 *

Cytokeratin 18

[16] BioMakor [22] BioMakor

RGE-53

Cytokeratin 18

[23] Eurodiagnostics

AS3- BIA2

Cytokeratin 19

[24] Progen

(Ks19.1) *

Kd.62

Cytokeratin 19

[25] Bio Makor

Ks19.2.105

[18] Progen

Vimentin V9*

Cytokeratin 19 Vimentin

Desmin *

Desmin

[27] BioMakor

NR4

Neurofilament 68-kDa

[28] Boehringer

GFAP

Glial fibrillary acidic protein

[28] BioMakor

D P i and

Desmoplakins

[29. 301 Progen

[26] BioMakor

DP2-2.15/2.17/2.19

Immunopcroxidase staining. This was performed on formalin-fixed paraffin-embedded as well as on frozen sections of tissues. For immunoperoxidase staining of paraffin-embedded material, 4-pm sections were deparaffinized and treated with 0.3% H 2 0 2in methanol for 30 min. After washing in phosphate-buffered saline (PBS; pH 7) and treating with normal blocking serum for 20 min at room temperature, sections were incubated with the primary antibodies for 30 min and with the diluted biotinylated secondary antibodies for 20 min both at room temperature and in a humid chamber. Bound antibodies were visualized by applying the avidin-biotin peroxidase complex (ABC) protocol [ 191 using the Vectastain, Elite, ABC Kit (Vector Lab, Burlingame, Ca, USA). For the localization of cytokeratins, desmin and neurofilament proteins the tissue sections were treated with 0.1% protease from Streptomyces griscus, type XIV (Sigma, St. Louis, Mo. USA) for 10-20 min at 37" C, prior to the immunochemical staining.

DG3.10

Desmoglein

a-SM actin * a-Smooth muscle actin

[31] Progen [32] BioMakor

* Antibodies used on formalin-fixed, paraffin-embedded sections ( ), Minor reactivity; The numbering of the various cytokeratin

polypeptides is according to Moll et al. [27]

For immunoperoxidase staining of frozen material, 4-pm-thick cryostat sections were fixed in acetone, air-dried. treated with normal blocking serum and incubated with the specific primary antibodies. Subsequent steps were similar to those used with the paraffin-embedded material except for treatment of slides with H,Oz after incubation with the secondary biotinylated antibodies.

244

Fig. 1A-F. Seminomas stained for intermediate filaments by the immunoperoxidase method on frozen and formalin-fixed paraflin-embedded matcrial. A Vimentin (V9) staining fibrous septae, blood vessels as well as scattered tumor cells (formalin-fixed, paraffin-embedded tissue, x 180). B Groups of seminoma cells staining for cytokerdtin 18 with antibody CY-90 (frozen tissue, x 360). C Diffuse distribution of cytokeratin 8 in seminoma, stained with antibody Ics8.17.2 (frozen tissue, x 180). D Seminoma cells staining with antibody CBlO for cytokeratin 4 (frozen tissuc, x 375). E Syncytiotrophoblastic giant cells in seminoma staining for cytokeratin 18 with antibody CY-90 (formalin-fixed, paraffin-embedded tissue, x 180). F Scattered seminoma cells staining positively with antibody to desmin (frozen tissue, x 375)

245

Results

Table 2. Intermediate filament expression in 26 human testicular seminomas

Of the 26 seminomas, 25 were of the classic type and one was spermatocytic. In 2 cases elements of embryonal carcinoma were present within the seminoma. Scattered syncytiotrophoblastic giant cells were seen in 3 seminomas.

No cytokeratins, no vimentin Vimentin only Cytokeratins only Vimentin and cytokeratins -

Immunolocalizarion microscopy

cytokeratins Neurofilament - In 2 of above cases: one with vimentin only and one with vimentin and cytokeratins

The immunohistochemical results obtained using immunofluorescence microscopy on frozen tissues, as well as immunoperoxidase staining on both frozen and formalin-fixed material are summarized in Table 2. In 4 seminomas the neoplastic cells showed no detectable IF proteins. In 3 cases, vimentin only was demonstrated in scattered tumor cells (Fig. 1 A). In 4 seminomas cy-

Desmin

4 cases 3 cases 4cases 15 cases

- In 3 of above cases with vimentin and

tokeratins were shown only in scattered cells, while in the remaining 15 cases both cytokeratins and vimentin were present in some cells, but in various proportions. Vimentin positivity in this group was mostly focal except in 6 cases in which it was widespread (Fig. 2).

Fig. 2. Widespread vimentin positivity in seminoma cells demonstrated by imrnunofluorescence. x 375

Fig. 3. Immunofluoresccnce staining of scattered seminoma cells with desmin antibody. x 500

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Fig. 4. Immunoperoxidase staining of scattered seminoma cells for neurofilament 68-kDa NF-L, on frozen tissue ( x 250). Insert shows immunofluorescence staining of same case for neurofilament NF-L x 280

Fig. 5. Diffuse punctuate immunofluorescence staining for desmoplakins in seminoma. x 375

Cytokeratins were present either in small distinct foci of tumor cells (Fig. 1 B) or, less commonly, more or less diffusely distributed either with a delicate but extensive fibril pattern (Fig. 1 C) or in sparse, plaques (not shown). The cytokeratins present were polypeptides 8, 18, (Fig. 1 B, C) as well as scant foci of 19 in five cases. In one tumor antibodies against cytokeratin 19 stained groups of cells. Coexpression of cytokeratins and vimentin, as studied by double-immunofluorescence staining, was present in scattered cells in six of these seminomas. In two tumors isolated cells showed an additional positive reaction for cytokeratin 4 (Fig. 1D) and, in another tumor, cytokeratin 17. Syncytiotrophoblastic giant cells within the seminomas stained strongly for cytokeratins 8, 18 and 19 (Fig. 1 E). One spermatocytic seminoma

studied showed scattered cells positive for cytokeratin 18. No other IFS were identified in this neoplasm. Desmin was expressed in seminoma cells in three instances. In one case desmin occurred in many cells, often showing an enrichment in a single juxtanuclear aggregate (Fig. 3), whereas in the other two cases only scattered tumor cells stained for desmin (Fig. 1 F). These cells were negative for a-SM actin, myosin and tropomyosin. In two other tumors some cells stained for 68kDa neurofilament protein (Fig. 4). Desmoplakins and desmoglein, usually scanty, were demonstrated in all cases in punctuate lines along the plasma membranes. Staining extent and intensity were somewhat variable (Fig. 5 ) . Stromal cells and blood vessels stained for vimentin

241

and a-smooth muscle actin and occasionally for desmin. Cytokeratins 8 and 18 decorated scattered cells in the stroma and in some instances blood vessel walls of these tumors. In three cases scattered stromal cells stained for cytokeratin 17.

Discussion Our observations that seminoma cells are capable of producing cytokeratins as well as vimentin confirm previous publications [9, 341. However, the extent and variety of I F proteins in tumor cells of our series far exceeded that reported in the past. Thus, in most instances, we found cytokeratins 8 and 18, although in some cases isolated tumor cells also expressed cytokeratin 19 in addition. Syncytiotrophoblastic giant cells stained strongly for cytokeratins 8, 18 and 19. Of particular interest was the occurrence of cytokeratin 4 in two tumors and cytokeratin 17 in one tumor. These observations indicate that the range of cytokeratin expression in seminomas is wider than hitherto suspected. Seminomas thus appear to possess not only a potential for differentiation of the simple epithelium type, but also, albeit limited, of the stratified epithelium type. The differentiation potential of seminomas, which is clearly higher than hitherto assumed, is also emphasized by the fact that in three cases tumor cells expressed desmin. Because of the presence of desmin in stromal smooth muscle cells and blood vessel cells throughout the tumor it was not feasible to verify our findings by gel electrophoresis. However, the use of four different monoclonal antibodies, all of which showed positive staining of seminoma cells, confirmed our results. It is of interest that the cells expressing desmin lacked skeletal muscle myosin, tropomysin and a-SM actin. This may be explained by the fact that desmin is the earliest known marker for cells in the myogenic lineage, while other muscle-specific proteins appear in later stages of myogenic differentiation [ 161, or may reflect spontaneous deregulation of the desmin gene in isolated cells of this tumor. In addition we noted neurofilament-protein-positive cells in two other cases. The presence of these unexpected antigens in seminoma cells should not come as a surprise, since it is known that both desmin [6, 131 and neurofilaments [13, 331 may appear in tissues lacking myogenic and neuronal derivation respectively [33]. In the case of germ cell tumors, this ability may be related to the local differentiation potential inherent in this tumor. From these and other observations it seems that the original concept [42] of seminoma as a very primitive germ cell neoplasm which has not acquired the potential for further differentiation, is no longer tenable. Particularly, the expression of nonepithelial cytoskeletal proteins such as desmin and neurofilaments in seminomas affirms the presence of pluripotent cells in this neoplasm. Our findings also support the concepts that “transitional forms” of germ cell tumors [17], and germ cell tumors with histology “borderline” between seminoma and embryonal carcinoma exist [47].

As reported by Denk et al. [9], we also observed positive staining of tumor cells, with antibodies to desmoplakins and desmoglein, albeit frequently scanty and irregular. These authors concluded that most desmosomes in seminomas are associated with IFS of the vimentin type, like certain other types of tumors containing cells with desmosomes but without cytokeratins, such as ovarian granulosa cell tumors [51 and meningiomas [371. Spermatocytic seminoma is a distinctive neoplasm as evidenced by its histology and biologic behaviour [41]. Its most likely origin is from relatively mature germ cells, namely spermatogonia. The one case contained in the present series showed cytokeratin 18 only in scattered tumor cells. Finally, the use of immunofluorescence microscopy on frozen tissues as well as immunoperoxidase staining of both frozen and formalin-fixed, paraffin-embedded material from the same tumors, led to a high degree of reliability. Each method used had its special advantages and disadvantages. Immunofluorescence microscopy of frozen tissue sections yielded highly reliable results, but interpreting the exact localization of the antibodyantigen reaction was more difficult. For the latter however, the immunoperoxidase methods on frozen tissue proved very valuable. Immunoperoxidase of formalinfixed, paraffin-embedded tissue was the easiest to interpret but here the panel of antibodies that could be used on formalin-fixed material was more restricted. Furthermore, because of the possible inactivating effect of formaldehyde, negative results were not always reliable. This was exemplified by one of the seminomas, which showed desmin in many cells by immunofluorescence and immunoperoxidase methods on frozen tissues, but failed to reveal this protein on formalin-fixed material. It can thus be concluded that labelling by antibodies should be performed in parallel with both frozen and fixed tissues, using immunofluorescence as well as immunoperoxidase methods. In conclusion, our results indicate that while some seminomas appear to be devoid of IF, most of them show varied differentiation patterns with epithelial or mesenchymal cell features, but occasionally also with myogenic and neurogenic ‘‘ markers”. These observations clearly classify seminomas as germ cell tumors capable of further differentiation, and may elucidate the relationship of seminoma to other germ cell tumors as well as helping us understand the histogenesis of these neoplasms. Acknowledgment. Supported by a grant from the Ministry of Science and Technology, Israel and the German Cancer Research Center (DKFZ), Heidelberg, FRG.

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