IMMUNOHISTOCHEMICAL GERM CELL NEOPLASIA E.

Rajpert-De Meyts and

University Department

IDENTIFICATION OF ANDROGEN

RECEPTORS

IN

N. E. Skakkeb\l=ae\k

of Growth and

Reproduction, Rigshospitalet, 9 Blegdamsvej,

ABSTRACT The highest prevalence of testicular cancer occurs in young men with high androgen activity. The presence and distribution of androgen receptors (ARs) was therefore investigated in germ cell neoplasia, using two specific monoclonal antibodies. Tissue samples from 18 patients with seminoma and/or carcinoma-in-situ (CIS) of the testis were examined. An indirect immunohistochemical method with a biotin-streptavidin-peroxidase or an alkaline phosphatase detection system was used. 45% of seminoma samples and 42% of CIS samples were AR\x=req-\ positive with antibody AN 1-15. The values obtained using antibody F 39.4.1 were 44 and 40% respectively. Some differences in specificity between the two antibodies were observed. Unusual granular staining of germ cells in normal testes, also present in malignant germ cells, was noted when antibody F 39.4.1 was used. The presence of AR protein immunoreactivity in neoplastic germ cells suggests that androgens may be involved in the pathogenesis of the disease.

DK-2100

Copenhagen,

Denmark.

been demonstrated in male reproductive organs, including the testis (Hansson, McLean, Smith et al. 1974). Within the testis, Leydig cells, Sertoli cells, and peritubular cells have been identified as AR positive, using recently developed specific antibodies (Sar, Lubahn, French & Wilson, 1990; Takeda, Chodak, Mutchnik et al. 1990). Normal germinal cells of the testis have been found to be negative for ARs, both by binding analysis (Grootegoed, Peters, Mulder et al. 1977) and by immunohistochemical staining (Sar et al. 1990; Takeda et al. 1990). There is little in the literature about ARs in testicular tumours and to our knowledge it is limited to radiolabelled ligand-binding assays (Bojar, Weissbach, Petzinna et al. 1985). The biochemical binding assays are sensitive, but they cannot provide information on cellular distribution, especially in cases such as CIS, where the malignant germ cells are scattered intratubularly among other tissue elements. We therefore undertook studies on the expression of ARs in germ cell malignancy using spe¬ cific monoclonal antibodies. -

MATERIALS AND METHODS

INTRODUCTION Germ cell cancer of the testis is mainly a disease of young men. It may occur immediately after puberty, although the peak incidence is found in males aged 25-45 years (Schot¬ tenfeld, Warshauer, Sherlock et al. 1980). Germ cell tumours, including seminomas and non-seminomas, are preceded by carcinoma-in-situ (CIS), which is a prein¬ vasive, intratubular abnormal germ cell pattern (see Plate, fig. 1). We have speculated that CIS germ cells are transformed gonocytes, the invasive growth of which depends on the pubertal activation of the pituitary-testis axis (Skakkebaek, Berthelsen, Giwercman & Müller, 1987). This hypothesis is in line with the observation that the rare cases of childhood germ cell cancer (yolk sac tumours) almost always occur in conjunction with the pre- and postnatal activation of the pituitary-testis axis (Winter & Faiman, 1972). However, there is almost no information on the role of hormones, including androgens, in the development of testicular cancer. Androgens are essential for male development and testicular maturation. Like other steroid hormones they

through specific intracellular receptors. Using radioli¬ gand binding analysis, androgen receptors (ARs) have

act

Antibodies and tissue samples. The monoclonal rat antihuman AR antibody AN 1-15 (Chang, Whelan, Popovich et al. 1989) was purchased from Affinity BioReagents (Neshanic Station, NJ, U.S.A.). The monoclonal mouse

antibody F 39.4.1 (Van Laar, VoorhorstOgink, Zegers et al. 1989; Zegers, Claassen, Neelen et al. 1991) was purchased from Sanbio (Uden, Holland). Parts anti-human AR

of testicular tumours and adjacent tissues were obtained from the department of pathology within minutes after surgery and snap-frozen at -80°C or fixed in 4% buffered formaldehyde or Stieve's solution and paraffin-embedded. Testicular tissue from 18 patients with seminoma and/or CIS were examined. Morphologically normal autopsy samples from suddenly deceased young men were used as controls. Frozen samples of prostate tissue removed from a patient with benign hyperplasia and fragments of epididymis removed during orchidectomy served as positive controls. The investigation was a part of a project approved by the local Ethical Committee. Immunohistochemistry. Frozen samples were sectioned at 5 µ , dry-mounted on poly-L-lysine-coated slides and fixed in 4% buffered formaldehyde or acetone. They were incubated overnight (4°C) with the primary antibodies at

the concentrations recommended by the suppliers. The remaining procedure was carried out at room tempera¬ ture. Between incubations the sections were washed in TBS, 1% (v/v) Triton X-100. Biotinylated goat anti-rat IgG (BioGenex, San Ramon, CA, U.S.A.) or rabbit antimouse IgG (Zymed, S. San Francisco, CA, U.S.A.) were used as secondary antibodies, followed by streptavidinperoxidase conjugate (DAKO, Glostrup, Denmark). To obtain brown staining, 3'3-diaminobenzidine tetrahydrochloride (DAB, Serva, Heidelberg, Germany) was used as a substrate (0.05% in TBS, 0.01% H202). Mounted sections were examined under a light microscope. For double staining with placental-like alkaline phosphatase (P1AP), a polyclonal rabbit anti-PlAP antibody (DAKO) was applied after developing AR staining with DAB. It was followed by alkaline phosphata¬ se (AP)-conjugated mouse anti-rabbit antibody The red colour was developed in 0.05 mol TBS/1, pH 8.7 containing 0.02% (w/v) sodium nitrate, 0.01% (v/v) new fuchsin, 0.05% (w/v) naphtol AS-bi-phosphate (Sigma, St levamisole/1 to block Louis, MO, U.S.A.) and 1 µ endogenous AP. Some slides were then lightly counterstained and mounted in aqueous medium. Paraffin-embedded sections were deparaffinized in petroleum and rehydrated in a series of alcohols and TBS. The procedure was then as described above for frozen samples, except that the sections fixed in Stieve's fixative were preincubated in 2.5 mg DNAse Fml (Shintaku & Said, 1987) and the formalin-fixed sections were preincubated in 0.1% (w/v) trypsin, 0.1% (w/v) CaCl2 (Andersen, 0rntoft & Poulsen, 1988) followed by 30 minincubation in DNAse I. Negative controls were included in all experi¬ ments. Adjacent sections were treated according to the procedure above, except that preimmune animal serum or a buffer were used instead of the primary antibody.

(DÀKO).

RESULTS

In frozen sections, positive staining for ARs appeared mainly in nuclei but a weak trace of cytoplasmic reaction was also observed when antibody AN 1-15 was used. In the patients with CIS, 5 out of 12 samples incubated with antibody AN-15 and 4 out of 10 samples incubated with F 39.4.1 showed nuclear staining of CIS germ cells (Plate, fig. 2 and 4). In seminoma samples the number of positive reactions was 5 out of 11 and 4 out of 9 respectively (Plate, fig. 3 and 5). The intensity of staining of malignant cells was in general weaker than that of Leydig cells but similar to that of Sertoli cells. The AR-positive cells usually showed varying intensity of staining within the same section. In several cases, but only when antibody F 39.4.1 was used, neoplastic germ cells exhibited unusual granular staining within their nuclei (Plate, fig. 4 and 5). The granules were also observed in two cases in which malignant cells were otherwise negative for ARs. Distinct single dots (usually one or two, rarely four) of positive

staining were tocytes in

also present in

spermatogonia and sperma(Plate,

two normal testes examined as controls

fig. 6).

The dots were present in frozen sections which fixed in acetone or formalin after cutting, but they were also observed in the sections immunostained for ARs without any fixation. Otherwise, normal germ cells were negative for ARs. As far as testicular somatic cells are concerned, nuclei of Leydig cells, peritubular cells, Sertoli cells and myoid cells of some vascular walls were immunoreactive with both antibodies in frozen sections. In paraffin-embedded sections, which we attempted to stain because they usually retain excellent morphology, strong nuclear immunoreactivity was observed in all sections examined, but only with antibody AN 1-15. were

However, all the testicular nuclei including lymphocytes stained. Moreover, Leydig cells, in contrast to the frozen sections, showed the weakest reaction. As this pattern suggested a possibility of a non-specific reaction,

were

therefore decided to exclude paraffin-embedded sections from the final results. Negative controls did not

we

exhibit any

staining.

DISCUSSION We report here the presence of AR protein immunore¬ in some cases of classical seminoma, the most common germ cell tumour and in CIS cells, an early preinvasive lesion. CIS cells were positive both when found in tissue adjacent to seminoma (two cases) and in five patients with exclusively CIS changes. To our know¬ ledge, there are no reports regarding immunohistochemi¬ cal studies of ARs in testicular tumours. In one study using a biochemical binding assay on testicular tumours (nine cases with various histology) no significant AR content was found (Bojar et al. 1985). Our results demon¬ strated nuclear immunoreactivity with two monoclonal antibodies, recognizing close but different epitopes of the human AR. Antibody F 39.4.1 was raised against a synthetic peptide corresponding to amino acids 301 320 (Van Laar et al. 1989); AN 1-15 was made against a fusion protein consisting of 242 amino acids starting from residue 331 (Chang et al. 1989). Several samples showed positive staining with only one of the two antibodies. This might indicate that the antibodies were recognizing a structurally modified AR or an AR-related protein. We do not know whether the reacting proteins are bioactive receptors or inactive variants. An involvement of other steroid hormones in the pathogenesis of cancer has been well established. Oestro¬ gen and progesterone receptors are important prognostic factors in breast cancer (Jensen, 1981). It has been suggested that an enhanced oestrogen and progesterone receptors state in a Leydig cell subpopulation is a basic pathophysiological factor in the development of Leydig cell tumours (Due, Dieckman, Loy & Stein, 1989). As far as androgens are concerned, the situation is less clear. An association between ARs and the development of pro-

activity

-

static carcinoma has been indicated, but it remains

disputed. The elucidation of the role of ARs in testicular cell proliferation is an even more complex issue, since, in the testis, the levels of androgens are very high and sex hormone-binding globulins known to interfere with androgen-mediated proliferative response are present (Damassa, Lin, Sonnenschein & Soto, 1991). According to our hypothesis, CIS may develop during fetal life. Verhoeven & Cailleau (1988) have shown that both androgens and follicle-stimulating hormone (FSH) are able to increase the concentration of

ARs in immature Sertoli cells and that their effects are additive. Testicular concentrations of testosterone, gonadotrophins and other factors involved in their regulation change during fetal development and testicular maturation (Buzek & Sanborn, 1988). It is thus possible to speculate that an imbalance in these complex interac¬ tions could be involved in the pathogenesis of germ cell

neoplasia.

An unusual and so far unreported finding is that several tumour samples as well as normal germ cells (except spermatozoa) showed granular staining of nuclei with antibody F 39.4.1. They were not detected when antibody AN 1-15 was used, which again showed the difference in specificity between the antibodies; and they were not present in negative controls. It is tempting to speculate that this observation may indicate the presence of a small number of saturated AR complexes tightly bound to DNA, which may be involved in the stimulation of certain specific transcripts linked to the neoplastic transformation. Because of the low number, possible change of protein conformation after DNA binding or an involvement of other factors, the complexes could escape detection by other methods. As F 39.4.1 antibody was raised against a synthetic oligopeptide, the domain it recognizes could remain unchanged; however, a reaction with an AR-related nuclear protein or even an artifact cannot be excluded. Further studies are required to establish the significance of this phenomenon. The presence of ARs in normal germ cells is controversial. Most authors believe that normal germ cells do not express functional AR (Grootegoed et al. 1977; Sar et al. 1990, Takeda et al. 1990), and that androgen action is mediated through some, as yet unknown factors produced by Sertoli cells. Hovewer, other reports suggest that androgens may have direct effects on germ cells (Wright & Frankel, 1980; Schulz, Paris, Lembke & Blüm, 1989), including a very recent report on the presence of AR mRNA in isolated spermatocytes and spermatids (Huang H.F.S et al. 1992, 74th Annual Meeting of the Endocrine Society, San Antonio, TX, U.S.A.). The posssible presence of steroid-responsive cell surface receptors in germ cells has also recently been disputed (Sheridan, 1991; Rommerts, 1992). In our study, we have demonstrated AR immunoreactivity in normal germ cells in the form of the single nuclear granules only. Authors reporting on ARs in male reproductive tissue have usually noted that the intensity of staining is

lower in the testis than in the prostate, seminal vesicle or In agreement with these results, the two antibodies that we studied gave stronger reaction in the prostate and the epididymis than in the testis, especially within the seminiferous tubules. The extremely high intratesticular concentration of androgens may cause significant down regulation of AR (Quarmby, Yarbrough, Lubahn et al. 1990). However, experimental studies on castrated animals showed a gradual decrease in immuno¬ histochemical detection of ARs after androgen withdrawal (Husmann, Wilson, McPhaul et al. 1990; Blok, Bartlett, Bolt-De Vries et al. 1992). According to Blok et al. (1992), those apparently contradictory observations could be explained by a structural modification of AR in the prolonged absence of androgens. Because of the generally weak intratubular AR staining and rather limited morphological resolution usually observed in frozen testicular tissue, in cases where there were problems we carried out double-staining using P1AP as a marker of CIS cells. This allowed us to make a much more reliable assessment and in fact resulted in a lower number of AR-positive CIS cases than initially diagnosed in a pilot study. Despite the inherent limitations of immunohisto¬ chemistry, our results should encourage further studies of AR expression in normal and neoplastic testicular germ cells using alternative methods such as in-situ hybridiza¬ tion. The presence of AR immunoreactivity in malignant germinal cells might indeed indicate that androgens are involved in the pathogenesis of testicular cancer.

epididymis.

ACKNOWLEDGEMENTS We wish to thank Drs G. Jenster and A. Brinkmann (Erasmus University Rotterdam, The Netherlands) for providing a sample of the F 31.4.1 antibody and Dr J.K Kristensen (Urology Department,

for help in obtaining samples of prostatic tissue. We thank Ms L. Andersen and Ms L. Cantell for performing cryostat sectioning. The study was supported by a grant from the Danish Cancer Society (No. 91-032).

Rigshospitalet)

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(1992). In Spermatogenesis Fertilization Contra¬ ception. Shering Foundation Workshop 4, pp. 1-19. Eds. E.Nieschlag & U.-F. Habenicht. Berlin: Springer-Verlag. Sar, M., Lubahn, D.B., French, F.S. & Wilson, E.M. (1990). Endocri¬ nology 126, 3180-3186. Rommerts, F.F.G.

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Schottenfcld, D., Warshauer, M.E., Sherlock, S., Zauber, A.G., Leder, M. & Payne, R. (1980). American Journal ofEpidemiology 111, 232-246. Schulz, R., Paris, F., Lembke, P. & Blüm, V. (1989). Journal of Histochemistry and Cytochemistry 37, 1667-1673. Sheridan, P.J. (1991). Molecular and Cellular Endocrinology 76, C39-

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C45.

DESCRIPTION OF PLATE FIGURE 1. Paraffin-embedded (Stieve's fixative) section of the testis with CIS stained with the anti-PlAP antibody. Only CIS cells (C) are stained with this marker, Sertoli cells (S) are negative. Germ cells in adjacent normal (N) tubules are negative. Bar = 100 µ .

FIGURE 4. Testis with CIS stained with the anti-AR antibody F 39.4.1 and with the anti-PlAP antibody. CIS cells (C) have dark PlAP-positive reaction in the cytoplasm and around their nuclei. Note the AR-positive granules in CIS cells nuclei (arrows). Bar 50 µ .

FIGURE 2. Frozen section of the testis with CIS stained with the anti-AR antibody AN 1-15. Note the AR-positive nuclei of CIS cells

FIGURE 5. Testis with Seminoma stained with antibody F 39.4.1. Note the AR-positive dots in the irregularly shaped tumour cells

(C) located along the basement membranes of seminiferous tubules. Sertoli (S), Leydig (L) and peritubular cells (P) are also positive. Bar = 100 µ .

FIGURE 3. Frozen section of the testis with Seminoma stained with antibody AN 1-15. Note AR-positive irregular nuclei of tumour cells. Bar

=

20 µ .

=

(arrows). Bar

=

50 µ .

FIGURE 6. Normal testis stained with antibody F 39.4.1. AR-positive Sertoli (S) and peritubular cells (P) are indicated. Note the ARpositive granules in unstained germ cells (arrows). Bar 50 µ . =