APMIS 99: 233-243, 1991
h t i n histochemistry of embryonal carcinoma RAIJA MALMI and KARL-OVE SODERSTROM Department of Pathology and Laboratory of Electron Microscopy, University of Turku, Turku, Finland
Malmi, R. & Soderstrom, K.-0. Lectin histochemistry of embryonal carcinoma. APMIS 99: 233-243, 1991. Histological tissue sections of human testicular embryonal carcinoma from 13 patients and of a xenograft tumour in nude mice, as well as cell lines of human embryonal carcinoma, were investigated with eight different lectins to characterize the distribution of glycoconjugates in embryonal carcinoma. In all cases the malignant cells showed binding with Con A, WGA and RCA I conjugates, whereas other lectins were bound to some, but never to all, tumour cells in each group, revealing the heterogeneity of the malignant cells. A polarization of cancer cells was shown particularly with WGA and RCA I labelling, which was most intense on the luminal borders of the carcinoma cells, where pseudotubular structures were formed. The sugar staining properties were retained in cell culture and in the xenograft tumour. Regardless of the germ cell origin, embryonal carcinoma cells differed from normal germ cells. The distribution of glycoconjugates was also different from that of testicular carcinoma-in-situ germ cells, which share morphological features and the pattern of glycosylation with seminoma cells. However, the similarities in lectin binding pattern of seminomas and embryonal carcinomas suggest the close relationship between the two types of testicular malignancy, without excluding the possibility that embryonal carcinomas were derived from seminomas. Although lectins seem to be less important for differential diagnostic use in testicular cancer, our findings showed the usefulness of lectin histochemistry for characterization of embryonal carcinoma. Key words: Lectins; glycoconjugates; embryonal carcinoma; testicular cancer; testis Raija Malmi, Department of Pathology, University of Turku, Kiinamyllynkatu 10, SF-20520 Turku, Finland
Carbohydrates, widely distributed throughout tissues at cell surfaces and within the cellular cytoplasm, are detectable by lectins which are able to distinguish different cell populations by their sugar expression (1 8). On the tissue level, lectin conjugates have been used as specific probes for detection of various cell types, as well as for detection of cells at various stages of differentiation or maturation. The lectins are valuable probes to explore changes associated with malignant transformation, metastasis, and tumour cell heterogeneity (1, 5). According to the present hypothesis, human germ cell neoplasia originates from the descendants of germ cells located in the gonads or extra-
Received May 7, 1990. Accepted August 27, 1990.
gonadal sites. However, the histogenesis of these tumours is not fully understood as there are not enough data about the premalignant stages, malignant transformation, or the sequence of events leading to invasive neoplasia ( 6 ) . Both seminomatous and non-seminomatous germ cell tumours of the testis are believed to originate from the intratubular carcinoma-in-situ (CIS) germ cells which have been observed in the testis antecedent to the appearance of various testicular germ cell tumours (29, 30). These carcinoma-in-situ germ cells are rich in glycogen and placental-like alkaline phosphatase (30). Whether embryonal carcinomas originate directly from transformed CIS gonocytes (30) or are derived from seminomas (27) is not clear, and additional evidence explaining the relationship between various germ cell tumours is needed (7). 233
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TABLE 1. Lectins used for the labellinn o f embrvonal carcinoma Lectin Abbreviation Major sugar specificity Binding inhibitor Canavalia ensiformis (jackbean) Con A a-D-Man; a-D-Glc, a-D-GlcNAc MM Triticum vulgaris (wheat germ) WGA (D-GlcNAc)2, NeuNAc GlcNAc Succinylated WGA S-WGA D-GlcNAc GlcNAc Ricinus communis (castor bean) RCA I a/P-D-Gal Gal Arachis hypogaea (peanut) PNA P-D-Gal( 1-3)-GalNAc Gal Glycine maximum (soybean) SBA alp-D-GalNAc, a-D-Gal GalNAc Helix pomatia H PA a-D-GalNAc GalNAc U I ~ europaeus X I UEA 1 a-L-Fucose Fucose Abbreviations used are Man, mannose; MM, a-methyl mannoside; Glc, glucose; GlcNAc, N-acetylglucosamine; NeuNAc, N-acetylneuraminic acid (sialic acid); Gal, galactose; GalNAc, N-acetylgalactosamine. For references see Goldstein & Hayes 1978 (12) and Bhavanandan & Katlic 1979 ( 2 ) .
TABLE 2. Lectin binding in embryonal carcinoma Lectin Patient Histological type") ConA WGA sWGA R C A I PNA VT embryonal carcinoma LK embryonal carcinoma +/HM embryonal carcinoma +/VJ embryonal carcinoma +/KH embr. carcinoma and teratoma ++ ++ +/SK embryonal carcinoma +/KJ embryonal carcinoma +/+/KJ embr. carcinoma, metastasis +/+/+/PH embr. carcinoma, metastask +/MV embr. carcinoma, infantile type ++ + +/VP embr. carcinoma and seminoma ++ ++ +/MP embryonal carcinoma +/AE embryonal carcinoma +I') According to WHO 1977 (26). + + = strong binding; + = moderate or faint binding; + / - = some focal binding; -
++ ++ ++ ++ ++ ++ ++ ++ ++ ++
Comparative studies have shown the similarity between carcinoma-in-situ germ cells and seminoma cells by means of immunohistochemistry and lectin histochemistry ( I I , 13, 22). Embryonal carcinoma cells are distinct from seminoma cells and have more features in common with early embryonic cells than with germ cells (6). In addition to the differences in lectin binding patterns between seminomas and embryonal carcinomas, the findings suggest that carbohydrate chains of human germ cell tumours
++ ++ ++ ++ ++ ++ ++
+
++ ++
+ ++ + ++ ++ ++ ++ ++ + + ++ ++ ++
SBA
HPA
-
-
+/-
-
-
-
-
-
-
-
+/+/-
-
+/=
-
+ -
no binding
change greatly in the process of differentiation from embryonal carcinoma cells to components of teratoid tumours or yolk sac carcinomas (32). In this work, both histological sections of Bouin- or formalin-fixed tissues from 13 patients with embryonal carcinoma of the testis, and histological sections of a xenograft tumour in nude mice, as well as three cell lines of human embryonal carcinoma, were treated with eight fluorescein-conjugated lectins of various carbohydrate binding specificities. The distribution
Fig. 1. Histological sections of human embryonal carcinoma fixed in Bouin's fluid. Phase contrast microphotographs of tumour (a) and (c), and corresponding areas labelled with FITC-Con A (b) and FITC-WGA (d). Notice the granular cytoplasmic staining reaction with FITC-Con A (b) in some of the cancer cells (arrows), distributed in a heterogenous manner. In addition to the less intense cytoplasmic staining, FITC-WGA (d) showed an intense fluorescence along free margins of cancer cells growing in a tubular pattern (arrows). Magnification (a, b) x 425; (c, d) x 270.
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and nature of cellular glycoconjugates of embryonal carcinoma are characterized. The staining results are compared with those of carcinomain-situ germ cells and seminoma cells of the testis to analyse the histogenetic differentiation of embryonal carcinoma related to the expression of carbohydrate moieties. MATERIALS AND METHODS Embryonal carcinoma Histological samples were collected from 13 human testicular embryonal carcinomas removed surgically at the Turku University Central Hospital during the years 1971-1987. The samples included two metastatic tumours. The WHO classification was used for the histological typing of the testis tumours (26). The specimens had been fixed in formalin for one or two days and were routinely processed and embedded in paraffin according to established histological procedures. In addition to the formalin-fixed tissue, frozen tissue and carcinoma tissue fixed in Bouin’s fluid directly after orchidectomy were available from two patients. Human embryonal carcinoma cell lines H 12.1, H 23.1.2 and H 1428A, and xenograft tumour tissue from nude mice were kindly provided by Dr C.Bokemeyer and Professor H . - J . Schmoll, the Medical University of Hannover, Hannover. West Germany. The properties of these lines have been discussed earlier (3, 4). The cells had been grown in monolayers on “Costar” coverslips in “Costar” bottles for two days, then fixed in 3.5% paraformaldehyde at 4°C for 10 min, and washed thoroughly in PBS before staining with lectins. In vitro the cell lines represent embryonal carcinoma with limited differentiation potential (4). The xenograft tumour tissue from the nude mice was fixed in formalin, embedded in paraffin, and histologically, a typical embryonal carcinoma was seen. Lectins Fluorescein isothiocyanate-conjugated (FITC) lectins (Vector Laboratories, Burlingame, CA) were used. The sources of lectins, their abbreviated names and nominal specificities are indicated in Table 1. For the staining experiments, all lectins were diluted in
PBS supplemented with C a t and Mg+ to contain 100-200 pg/ml of the lectin conjugate. The specificity of the staining obtained with each lectin was tested by preincubating the lectin in a 0.2 M solution of the appropriate inhibitory saccharide before the staining experiments. +
+
Staining procedures 5 pm thick sections were cut from the paraffinembedded material. The sections were then deparaffnized in graded alcohols and transferred into PBS, pH 7.4. The labelling was done by applying a drop of the diluted lectin solution on the sections and on the’coverslips with monolayers of human embryonal carcinoma cells. These were then placed in a moist chamber and incubated with the lectins for 30 min. The lectin solutions were removed by washing the sections and the coverslips three times in PBS and twice in distilled water. The slides and coverslips were mounted with Fluoromount (Gurr BDH Chemicals, England) and observed with a Leitz epifluorescence microscope with the appropriate filter system for FITC fluorescence.
RESULTS
Embryonal carcinoma f r o m patients with trsticular cancer
The binding patterns of lectins in the 13 formalin-fixed testicular tumours removed surgically are summarized in Table 2. In all cases, histology typical for embryonal carcinoma was found. All tumour cells were positive for Con A, WGA and RCA 1. Less uniform binding of some carcinoma cells was observed by s-WGA in six cases, by PNA in ten cases, by SBA in four cases, and by HPA in one case. Since UEA I reactivity was characterized as dull and unclear, this lectin was excluded from Table 2. Since the binding of lectins in testicular tissue was influenced by the fixation method used (23), we compared the labelling results of the two testicular tumours (VP, MP) from which Bouinfixed tissue was available with those fixed in formalin. In general, the intensity of fluor-
~
Fig. 2. Histological sections of human embryonal carcinoma fixed in Bouin’s fluid. Phase contrast microphotographs of tumour (a) and (c), and corresponding areas stained with FITC-RCA 1 (b) and FITC-PNA (d). FITC-RCA I (b) labelled the cell borders and cytoplasm of carcinoma cells. The apical surfaces of cancer cells forming tubule-like structures showed particularly bright fluorescence (arrows). FITC-PNA (d) was positive in focal regions of carcinoma tissue. The labelling reaction was located along free borders of carcinoma cells (arrows), forming papillary structures. Magnification (a, b, c, d) x 425.
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escence was somewhat weaker in the formaldehyde-fixed than in the Bouin-fixed specimens, but slight changes in the staining pattern, depending on the fixative, were also observed. Parallel with paraffin-embedded material, acetonefixed frozen sections of the tumours were also used. Due to the poorly preserved morphology of such tumour cells, their staining results were not considered reliable. FITC-Con A was brightly bound to the cytoplasm of all the embryonal carcinoma cells; the fluorescence frequently appeared in a granular pattern (Figs. la, b). A similar heterogeneous distribution of granular reactivity among cancer cells was observed in tissues fixed in formalin or in Bouin. FITC-WGA labelled the cytoplasm of all tumour cells with an intense reaction along their luminal borders (Figs. lc, d) under both fixation conditions. In contrast, FITC-s-WGA showed weak staining of some tumour cells in the Bouinfixed specimens and was negative in the corresponding formalin-fixed tissues. FITC-RCA I demonstrated bright labelling of carcinoma cells, with a more pronounced staining on apical surfaces of the malignant cells (Figs. 2a, b). Some decrease in the intensity of the labelling reaction was observed in formalinfixed tissue samples. FITC-PNA was positive in the two Bouinfixed tumours. Fluorescence of some carcinoma cells was predominantly visualized on free cellular margins (Figs. 2c, d). The intensity of labelling was decreased in formalin-fixed tumours. FITC-SBA showed heterogeneous reactivity of some carcinoma cells in histological sections of the two Bouin-fixed tumours, while it was hardly positive in formalin-fixed tissues. FITCUEA I was also faintly positive when Bouin’s fluid was used for tissue fixation, but no clear staining was detected in formalin-fixed tissue.
FITC-HPA was negative in the carcinoma cells of the two tumours regardless of the fixative used. A few tubules with CIS germ cells were found in the periphery of Bouin-fixed (MP) and formalin-fixed (VT, HM, KH, SK, VP, MP) tissues of six testicular tumours. In all cases, Con A showed strong, often granular, reactivity in the cytoplasm of CIS cells. A less intense labelling of CIS germ cells was also achieved with WGA in all these tumours. RCA I was bound to all CIS cells in the Bouin-fixed tissue of the tumour (MP) and to some CIS cells in two formalinfixed tumours (VT, KH). S-WGA, PNA, SBA and HPA did not label CIS germ cells present adjacent to the Bouin- and formalin-fixed tumour tissue of these six embryonal carcinomas. The small seminomatous component of the tumour (VP) was strongly labelled with Con A. WGA and RCA I were also bound to the seminomatous cells of this tumour. Weak binding of s-WGA to some of the cells in the seminomatous tumour component was observed. PNA, SBA and HPA did not clearly label the seminoma cells of the combined tumour. Lectin binding properties of the stromal connective tissue and of the inflammatory cells infiltrating the tumours are not reported here as the main interest of the present study was to characterize the nature of embryonal carcinoma cells. In the control experiments, addition of inhibitory sugars to the lectin solutions resulted in either absence of or significant reduction in binding of the lectins. Xenograft tumour The xenograft tumour in nude mice, histologically representing embryonal carcinoma (Fig. 3a), showed a bright granular labelling of carcinoma cells by Con A (Fig. 3b). Malignant cells
Fig. 3. Histological sections of embryonal carcinoma implanted in nude mouse and stained with haematoxylin and eosin (a), FITC-Con A (b), FITC-WGA (c) and FITC-RCA I (d). (a) Embryonal carcinoma cells with large atypical nuclei (arrows) showed abundant mitotic figures. The general pattern of embryonal carcinoma was preserved in the transplant. FITC-Con A (b) showed a cytoplasmic labelling reaction which was granular in the majority of the malignant cells (arrows). FITC-WGA (c) was evenly bound to the cytoplasm of carcinoma cells (arrows), but labelling was more intense on free borders of cancer cells. FITC-RCA I (d) labelled cell borders brightly with a more prominent fluorescence along luminal margins of tubular structures (arrows). Magnification (a, b) x 350; (c, d) x 425.
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were also labelled by WGA and RCA I with an intense fluorescence along apical borders of tumour cells (Figs. 3c, d), forming small tubular structures. Some faint reactivity was observed by s-WGA. The tumour cells were stained by PNA, displaying a linear fluorescence of free cellular margins. For SBA and UEA I, dull and faint fluorescence of the carcinoma was seen. HPA did not label the murine carcinoma cells at all. Embryonul carcinoma cells in vitro Three cell lines of human embryonal carcinoma H 12.1, H 23.1.2, and 1428 A displayed typical morphological characteristics of embryonal carcinoma cells (Fig. 4a). Various cell lines were stained by Con A, which presented cytoplasmic fluorescence that, in some of the cells, was granular (Fig. 4b). The positive labelling reaction by WGA in all three cell lines was located especially along cell surfaces and in the perinuclear region of the cultured cells (Fig. 4c). S-WGA was weakly bound to the cytoplasm of carcinoma cells in all three cell lines. The cell lines were also stained by RCA I with a labelling reaction in the cytoplasm and along cell borders of embryonal carcinoma cells. PNA was faintly positive, whereas SBA did not clearly label malignant cells. Various cell lines failed to react with HPA. On the other hand, some dull intracellular labelling was noted by UEA I. DISCUSSION Using a panel of lectins we found histochemically that embryonal carcinoma cells from both patients with testicular cancer as well as experimental models were positively labelled by Con A, WGA, and RCA I. Other lectins showed focal and less uniform binding patterns of the malignant cells. In tumour cells, the presence of a great number of mannose- and glucose-containing glycoconjugates is demonstrated by positive cytoplasmic reactivity with Con A, as the lectin binds to polymannosyl cores of carbohydrate chains (20). The intensely granular Con A labelling in a portion of the malignant cells of each tumour suggests the heterogeneous nature of the tumour cell population. The accumulation of carbohydrate-rich substances in the cytoplasm may 240
Fig. 4. Embryonal carcinoma cells grown in a monolayer (H l2.l), fixed in 3.5% paraformaldehyde. Phase contrast microphotograph (a) and cells stained with FITC-Con A (b) and FITC-WGA (c). FITCCon A (b) was bound in a granular way to the cytoplasm of most of the tumour cells (arrows), whereas FITC-WGA (c) showed a more uniform labelling of cultivated cells (arrows). Magnification (a, b) x 425; (c) x 270.
partially be explained by defective membrane incorporation or secretion of glycoconjugates, as reported for other malignancies (1 7). Further explanations may also include the accelerated
LECTINS IN TESTICULAR CANCER
production of glycosylated proteins, such as alpha-fetoprotein and human chorionic gonadotrophin, which are the most important tumour markers of non-seminomatous germ cell tumours and are known to interact with Con A (15, 16). In addition to the less intense cytoplasmic staining, WGA binding sites were predominantly located on the luminal surfaces of carcinoma cells, forming tubular structures. This indicates a polarization of the embryonal carcinoma cells which was lost during cell culture, and which thus may be due to stromal factors having an influence on cell polarity. Similar polarity has also been found in mammary carcinoma with the apocrine membrane antigen, which sometimes also seems to persist in cell culture (10). WGA specifically binds sialic acid and N-acetylglucosamine (2, 12). In contrast, its succinylated derivative, s-WGA, is specific for N-acetylglucosamine only (25). As s-WGA was faintly positive or in many cases negative in different tumours, the bright fluorescence by WGA was apparently due to the high content of sialic acid in embryonal carcinoma cells. A similar increase in the sialic acid content is reported for many other carcinomas e.g. in breast (31), kidney (9), and colon (14). Lectin binding polarity of the tumour cells was also demonstrated by RCA I, suggesting the presence of terminal galactose and galactose residues at the penultimate position when substituted by sialic acids (8). Another galactose-specific lectin PNA, with a more limited binding specificity (12), focally labelled part of the embryonal carcinoma cells in both the Bouin-fixed tumours and in 10 formalin-fixed testicular tumours. The heterogeneous labelling of embryonal carcinoma cells observed with PNA apparently reflects either functionally or differentially related characteristics of individual cells, substantiating the results reported by other investigators (28, 32). On the other hand, PNA was more sensitive to the fixation method used in this study than were Con A, WGA, s-WGA, and RCA I. The lectin staining is clearly decreased when formaldehyde is used as fixative (23). Embryonal carcinoma cells showed heterogeneous reactivity to SBA, HPA and UEA I which was not dependent on the possible combination of other histological types with embry-
onal carcinoma. SBA recognizes N-acetylgalactosamine residues (12). Only four tumours presented some positive foci with this lectin. Another N-acetylgalactosamine-specific (1 2) lectin HPA was negative in almost all cases; one formalin-fixed tumour (JK), derived from undescended testis and with an aggressive clinical behaviour, reacted positively to HPA. The functional significance of this observation remains speculative, as a systematic correlation of lectin binding with the clinical data of the testicular cancer patients was not done during the study. The fucose-specific UEA I presented an unclear and dull reaction in histological sections of formalin-fixed tumours, yet it was positive in the Bouin-fixed specimens. Detection of the small amount of fucose found in some of the embryonal carcinomas was not verified only by the sensitivity to the fixative used (23), but is also reported by other investigators (32). The heterogeneous nature of embryonal carcinomas with respect to their sugar composition has also been shown by antibodies, e.g. by the anti-1(Ma) antibody which recognizes a branched carbohydrate chain composed of three N-acetyllactosamine units (32). Regardless of the germ cell origin, the expression of glucosyl moieties of embryonal carcinomas differed from that of normal germ cells ' during spermatogenic cell differentiation (2 1). When the lectin binding pattern of embryonal carcinomas is compared with that of testicular CIS and seminoma cells (21,22,24), the positive labelling of all varying cell types by Con A, WGA and RCA I suggests that the neoplastic cells are closely related. However, the specific differences in the distribution of binding sites by each lectin observed during this study and verified in the previous studies (21, 22, 32) illustrate that the nature of embryonal carcinomas is distinct from that of seminomas. The different features of embryonal carcinoma cells in comparison with seminoma cells are also reflected by PNA reactivity, as the majority of classic seminomas are negative for this lectin (19, 22, 24). In our study, even most of the formalinfixed tumours presented heterogeneous focal PNA reactivity, located on free cellular margins, adding evidence to the suggestion that the appearance of this marker is differentiation related (32). On the other hand, CIS germ cells and seminoma cells seem to have morphological fea24 1
MALMI & St)DERSTRt)M
tures (30) and patterns of glycosylation (22) in common. Therefore, the lectin histochemical results do not exclude the model of tumour pathogenesis in which embryonal carcinomas develop through a seminoma stage from intratubular CIS germ cells (7, 27). Otherwise, the regression of CIS germ cells into totipotent embryonal carcinoma cells (30) must be associated with distinct alterations in the distribution of cellular glycoconjugates. In the human germ cell tumour lines established to provide a model for the study of the differentiation of human germ cell tumours in vivo, the expression of Con A, WGA, RCA I and PNA binding sites was observed. The lectin staining pattern in histological sections of the xenograft tumour did not differ significantly from that of human embryonal carcinoma from testicular cancer patients. Our findings speak in favour of the usefulness of cell culture and animal models in studies of germ cell tumours. The results presented show that detection of cellular carbohydrates by lectin histochemistry is of potential significance in the investigation of the nature and origin of human embryonal carcinoma, both in histological sections and in experimental models. The use of lectins, in combination with other cellular markers and clinical data, provides an additional analytical tool for the study of human germ cell tumours. The skilful technical assistance of Mrs A . Vuoristo and Mr J. Liippo is kindly acknowledged. We thank Dr. C . Bokemeyer and Prof. H.-J. Schmoll for providing us with the human germ cell lines and the murine xenograft tumour. This study was supported by the Cancer Society and the Academy of Finland.
REFERENCES Ahoy, J., Ucci, A . A . & Pereira, M . E.: Lectin Histochemistry: An update. In: DeLellis, R . A . (Ed.): Advances in Immunohistochemistry, Raven Press, New York, 93-131, 1988. Bhavanandan, K & Katlic, A.: The interaction of wheat germ agglutinin with sialoglycoproteins the role of sialic acid. J. Biol. Chem. 254: 400W008, 1979. Casper, J., Schmoll. H.-J., Schnaidt, U. & Fonatsch, C.: Cell lines of human germinal cancer. Int. J. Androl. 10: 105-113, 1987. Casper, J . , Bronson, D. L., Harstrick, A . , Schnaidt, U. & Schmoll, H.-J.: Growth of human
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germ cell tumour cell lines in nude mice. In: Abstract book: Pathobiology of human germ cell neoplasia. Satellite Meeting of the 1 1 th International Congress of the International Society of Developmental Biologists, 1989 (abstr). 5. Damjanov, I.: Biology of disease. Lectin cytochemistry and histochemistry. Lab. Invest. 57: 5-20. 1987. 6. Damjanov, I.: Developmental biology of human germ cell tumours. In: Abstract book: Pathobiology of human germ cell neoplasia. Satellite Meeting of the 11th International Congress of the International Society of Developmental Biologists, 1989 (abstr). 7. Damjanov, I.: Is seminoma a relative or a precursor of embryonal carcinoma'? Lab. Invest. 60: 1-3, 1989. 8. Debray, H . . Decout, D., Strecker, G., Spik, G. & Montreul, J.: Specificity of twelve lectins towards oligosaccharides and glycopeptides related to Nglycosylproteins. Eur. J. Biochem. f f7: 41-55, 1981. 9. Echenique, J. E . & Graham S. D.: Significance of lipid-associated sialic acid and CA 19-9 as tumor markers for renal cell carcinoma. Urology 32: 397400, 1988. 10. Forsman, L.: An apocrine membrane antigen with polarized distribution and hormonally regulated expression in human endometrial and mammary carcinoma cell lines. APMIS 95: 315-323, 1987. 11. Giwercman, A , , Marks, A . , Bailey, D., Baumal, R. & Skakkehaek, N . E.: A monoclonal antibody as a marker for carcinoma in situ germ cells of the human adult testis. APMIS 96: 667-670, 1988. 12. Goldstein, I. J. & Hayes, C . E.: The lectins: Carbohydrate binding proteins of plants and animals. Adv. Carbohydr. Chem. Biochem. 35: 127-340, 1978. 13. Hustin, J., Collette, J . & Franchimont, I?: Immunohistochemical demonstration of placental alkaline phosphatase in various states of testicular development and in germ cell tumours. Int. J. Androl. 10: 29-35, 1987. 14. Irimura, i?, Carlson, D. A , , Price. J., Yumori, T , Giavazzi, R . , Ota, D. M . & Cleary, K . R.: Differential expression of a sialoglycoprotein with an approximate molecular weight of 900,000 on metastatic human colon carcinoma cells growing in culture and in tumor tissues. Cancer Res. 48: 2353-2360, 1988. 15. Ishiguro, T , Sawada, M . , Sakaguchi, H . & Sugitachi, I.: Alpha-fetoprotein subfractions in germ cell tumours identified by Con A or LCH crossedline affinity immunoelectrophoresis. AJRI 5: 102-105, 1984. 16. Koyama, K., Toda, K . , Kuriyama, D. & Isojima, S.: Affinity to lectin, biological and immunological characteristics of human chorionic gonado-
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17.
18.
19.
20.
tropins from pregnant women and trophoblastic tumour patients. Acta Endocrinol. 112: 579-585, 1986. Langkilde, N . C., WolJ H . & Orntoft, T E: Lectinhistochemistry of human bladder cancer: Loss of lectin binding structures in invasive carcinomas. APMIS 97: 367-373, 1989. Leathern, A.: Lectin histochemistry. In: Polak, J. M . , van Noorden, S. (Eds): Immunocytochemistry. Modern Methods and Applications. Second Edition. 167-187, 1986. Lee, M.-C., Talerman, A . , Oosterhuis, J. W & Damjanov, I.; Lectin histochemistry of classic and spermatocytic seminoma. Arch. Pathol. Lab. Med. 109: 938-942, 1985. Lotan. R . & Nicholson. G.: Purification of cell membrane glycoproteins by lectin affinity chromatography. Biochem. Biophys. Acta 559: 7 7 0 77c, 1n-m
J L 7 - J / U I 1717.
21. Malmi, R. & Soderstrom, K.-0.: Lectin binding sites in human seminiferous epithelium, in CIS cells and in seminoma. Int. J. Androl. 10: 157-162, 1987. 22. Malmi, R . & Soderstrom, K.-0.: Lectin binding to carcinoma-in-situ cells of the testis. A comparative study of CIS germ cells and seminoma Virchows Arch. A Anat) 413: 69-75, 1988. 23. Malmi, R. & Soderstrom, K.-0.: Lectin binding to rat spermatogenic cells: effects of different fixation methods and proteolytic enzyme treatment. Histochem. J. 20; 276282, 1988. 24. Malmi, R.. Kellokumpu-Lehtinen, I?-L. & Siiderstrom, K.-0.: Correlation between lectin binding and clinical factors in seminoma patients. J. Cancer Res. Clin. Oncol. 115: 96100, 1989.
25. Monsigny, M . . Sene, C . , Ohrenovitch, A , , Roche, A.-C., Delmotte, E & Boschetti, E: Properties of succinylated wheat germ agglutinin. Eur. J. Biochem. 98: 3945, 1979. 26. Mostofi, E K . & Sobin, L. H.: Histological typing of testis tumours. World Health Organization, Geneva, 1977. 27. Oosterhuis, J . W , Castedo, S. M . M . J . , de Jong, B., Cornelisse, C. J . , Dam, A . , Sleijyer, D. 7: & Schraffordt Koops, H.: Ploidy of primary germ cell tumours of the testis: pathogenetic and clinical relevance. Lab. Invest. 60: 14-21, 1989. 28. Reisner, Y , Gachelin, G . , Dubois, I?, Nicolas, J.-E, Sharon, N . & Jacob, F : Interaction of peanut agglutinin, a lectin specific for nonreducing terminal D-galactosyl residues, with embryonal carcinoma cells. Dev. Biol. 61: 2C27, 1977. 29. Skakkebaek, N . E.: Carcinoma-in-situ of the testis: frequency and relationship to invasive germ cell tumours in infertile men. Histopathol. 2: 157-170, 1978. 30. Skakkehaek, N . E . , Berthelsen, J . G . , Giwercman, A . & Miiller, J.: Carcinoma-in-situ of the testis: possible origin from gonocytes and precursor of all types of germ cell tumours except spermatocytoma. Int. J. Androl. 10: 19-28, 1987. 31. Stahli, C . , Caravatti, M . , Aeschbacher, M . , Kocyba, C.. Takacs, B. & Carmann, H.: Mucin-like carcinoma-associated antigen defined by three monoclonal antibodies against different epitopes. Cancer Res. 48: 6799-6802, 1988. 32. Teshima, s., Hirohashi, s., Shimosato, Z, Kishi, K . , Ino, Z, Matsumoto, K . & Yamada, T : Histochemically demonstrable changes in cell surface carbohydrates of human germ cell tumors. Lab. Invest. 50; 271-277, 1984.
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h t i n histochemistry of embryonal carcinoma RAIJA MALMI and KARL-OVE SODERSTROM Department of Pathology and Laboratory of Electron Microscopy, University of Turku, Turku, Finland
Malmi, R. & Soderstrom, K.-0. Lectin histochemistry of embryonal carcinoma. APMIS 99: 233-243, 1991. Histological tissue sections of human testicular embryonal carcinoma from 13 patients and of a xenograft tumour in nude mice, as well as cell lines of human embryonal carcinoma, were investigated with eight different lectins to characterize the distribution of glycoconjugates in embryonal carcinoma. In all cases the malignant cells showed binding with Con A, WGA and RCA I conjugates, whereas other lectins were bound to some, but never to all, tumour cells in each group, revealing the heterogeneity of the malignant cells. A polarization of cancer cells was shown particularly with WGA and RCA I labelling, which was most intense on the luminal borders of the carcinoma cells, where pseudotubular structures were formed. The sugar staining properties were retained in cell culture and in the xenograft tumour. Regardless of the germ cell origin, embryonal carcinoma cells differed from normal germ cells. The distribution of glycoconjugates was also different from that of testicular carcinoma-in-situ germ cells, which share morphological features and the pattern of glycosylation with seminoma cells. However, the similarities in lectin binding pattern of seminomas and embryonal carcinomas suggest the close relationship between the two types of testicular malignancy, without excluding the possibility that embryonal carcinomas were derived from seminomas. Although lectins seem to be less important for differential diagnostic use in testicular cancer, our findings showed the usefulness of lectin histochemistry for characterization of embryonal carcinoma. Key words: Lectins; glycoconjugates; embryonal carcinoma; testicular cancer; testis Raija Malmi, Department of Pathology, University of Turku, Kiinamyllynkatu 10, SF-20520 Turku, Finland
Carbohydrates, widely distributed throughout tissues at cell surfaces and within the cellular cytoplasm, are detectable by lectins which are able to distinguish different cell populations by their sugar expression (1 8). On the tissue level, lectin conjugates have been used as specific probes for detection of various cell types, as well as for detection of cells at various stages of differentiation or maturation. The lectins are valuable probes to explore changes associated with malignant transformation, metastasis, and tumour cell heterogeneity (1, 5). According to the present hypothesis, human germ cell neoplasia originates from the descendants of germ cells located in the gonads or extra-
Received May 7, 1990. Accepted August 27, 1990.
gonadal sites. However, the histogenesis of these tumours is not fully understood as there are not enough data about the premalignant stages, malignant transformation, or the sequence of events leading to invasive neoplasia ( 6 ) . Both seminomatous and non-seminomatous germ cell tumours of the testis are believed to originate from the intratubular carcinoma-in-situ (CIS) germ cells which have been observed in the testis antecedent to the appearance of various testicular germ cell tumours (29, 30). These carcinoma-in-situ germ cells are rich in glycogen and placental-like alkaline phosphatase (30). Whether embryonal carcinomas originate directly from transformed CIS gonocytes (30) or are derived from seminomas (27) is not clear, and additional evidence explaining the relationship between various germ cell tumours is needed (7). 233
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TABLE 1. Lectins used for the labellinn o f embrvonal carcinoma Lectin Abbreviation Major sugar specificity Binding inhibitor Canavalia ensiformis (jackbean) Con A a-D-Man; a-D-Glc, a-D-GlcNAc MM Triticum vulgaris (wheat germ) WGA (D-GlcNAc)2, NeuNAc GlcNAc Succinylated WGA S-WGA D-GlcNAc GlcNAc Ricinus communis (castor bean) RCA I a/P-D-Gal Gal Arachis hypogaea (peanut) PNA P-D-Gal( 1-3)-GalNAc Gal Glycine maximum (soybean) SBA alp-D-GalNAc, a-D-Gal GalNAc Helix pomatia H PA a-D-GalNAc GalNAc U I ~ europaeus X I UEA 1 a-L-Fucose Fucose Abbreviations used are Man, mannose; MM, a-methyl mannoside; Glc, glucose; GlcNAc, N-acetylglucosamine; NeuNAc, N-acetylneuraminic acid (sialic acid); Gal, galactose; GalNAc, N-acetylgalactosamine. For references see Goldstein & Hayes 1978 (12) and Bhavanandan & Katlic 1979 ( 2 ) .
TABLE 2. Lectin binding in embryonal carcinoma Lectin Patient Histological type") ConA WGA sWGA R C A I PNA VT embryonal carcinoma LK embryonal carcinoma +/HM embryonal carcinoma +/VJ embryonal carcinoma +/KH embr. carcinoma and teratoma ++ ++ +/SK embryonal carcinoma +/KJ embryonal carcinoma +/+/KJ embr. carcinoma, metastasis +/+/+/PH embr. carcinoma, metastask +/MV embr. carcinoma, infantile type ++ + +/VP embr. carcinoma and seminoma ++ ++ +/MP embryonal carcinoma +/AE embryonal carcinoma +I') According to WHO 1977 (26). + + = strong binding; + = moderate or faint binding; + / - = some focal binding; -
++ ++ ++ ++ ++ ++ ++ ++ ++ ++
Comparative studies have shown the similarity between carcinoma-in-situ germ cells and seminoma cells by means of immunohistochemistry and lectin histochemistry ( I I , 13, 22). Embryonal carcinoma cells are distinct from seminoma cells and have more features in common with early embryonic cells than with germ cells (6). In addition to the differences in lectin binding patterns between seminomas and embryonal carcinomas, the findings suggest that carbohydrate chains of human germ cell tumours
++ ++ ++ ++ ++ ++ ++
+
++ ++
+ ++ + ++ ++ ++ ++ ++ + + ++ ++ ++
SBA
HPA
-
-
+/-
-
-
-
-
-
-
-
+/+/-
-
+/=
-
+ -
no binding
change greatly in the process of differentiation from embryonal carcinoma cells to components of teratoid tumours or yolk sac carcinomas (32). In this work, both histological sections of Bouin- or formalin-fixed tissues from 13 patients with embryonal carcinoma of the testis, and histological sections of a xenograft tumour in nude mice, as well as three cell lines of human embryonal carcinoma, were treated with eight fluorescein-conjugated lectins of various carbohydrate binding specificities. The distribution
Fig. 1. Histological sections of human embryonal carcinoma fixed in Bouin's fluid. Phase contrast microphotographs of tumour (a) and (c), and corresponding areas labelled with FITC-Con A (b) and FITC-WGA (d). Notice the granular cytoplasmic staining reaction with FITC-Con A (b) in some of the cancer cells (arrows), distributed in a heterogenous manner. In addition to the less intense cytoplasmic staining, FITC-WGA (d) showed an intense fluorescence along free margins of cancer cells growing in a tubular pattern (arrows). Magnification (a, b) x 425; (c, d) x 270.
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and nature of cellular glycoconjugates of embryonal carcinoma are characterized. The staining results are compared with those of carcinomain-situ germ cells and seminoma cells of the testis to analyse the histogenetic differentiation of embryonal carcinoma related to the expression of carbohydrate moieties. MATERIALS AND METHODS Embryonal carcinoma Histological samples were collected from 13 human testicular embryonal carcinomas removed surgically at the Turku University Central Hospital during the years 1971-1987. The samples included two metastatic tumours. The WHO classification was used for the histological typing of the testis tumours (26). The specimens had been fixed in formalin for one or two days and were routinely processed and embedded in paraffin according to established histological procedures. In addition to the formalin-fixed tissue, frozen tissue and carcinoma tissue fixed in Bouin’s fluid directly after orchidectomy were available from two patients. Human embryonal carcinoma cell lines H 12.1, H 23.1.2 and H 1428A, and xenograft tumour tissue from nude mice were kindly provided by Dr C.Bokemeyer and Professor H . - J . Schmoll, the Medical University of Hannover, Hannover. West Germany. The properties of these lines have been discussed earlier (3, 4). The cells had been grown in monolayers on “Costar” coverslips in “Costar” bottles for two days, then fixed in 3.5% paraformaldehyde at 4°C for 10 min, and washed thoroughly in PBS before staining with lectins. In vitro the cell lines represent embryonal carcinoma with limited differentiation potential (4). The xenograft tumour tissue from the nude mice was fixed in formalin, embedded in paraffin, and histologically, a typical embryonal carcinoma was seen. Lectins Fluorescein isothiocyanate-conjugated (FITC) lectins (Vector Laboratories, Burlingame, CA) were used. The sources of lectins, their abbreviated names and nominal specificities are indicated in Table 1. For the staining experiments, all lectins were diluted in
PBS supplemented with C a t and Mg+ to contain 100-200 pg/ml of the lectin conjugate. The specificity of the staining obtained with each lectin was tested by preincubating the lectin in a 0.2 M solution of the appropriate inhibitory saccharide before the staining experiments. +
+
Staining procedures 5 pm thick sections were cut from the paraffinembedded material. The sections were then deparaffnized in graded alcohols and transferred into PBS, pH 7.4. The labelling was done by applying a drop of the diluted lectin solution on the sections and on the’coverslips with monolayers of human embryonal carcinoma cells. These were then placed in a moist chamber and incubated with the lectins for 30 min. The lectin solutions were removed by washing the sections and the coverslips three times in PBS and twice in distilled water. The slides and coverslips were mounted with Fluoromount (Gurr BDH Chemicals, England) and observed with a Leitz epifluorescence microscope with the appropriate filter system for FITC fluorescence.
RESULTS
Embryonal carcinoma f r o m patients with trsticular cancer
The binding patterns of lectins in the 13 formalin-fixed testicular tumours removed surgically are summarized in Table 2. In all cases, histology typical for embryonal carcinoma was found. All tumour cells were positive for Con A, WGA and RCA 1. Less uniform binding of some carcinoma cells was observed by s-WGA in six cases, by PNA in ten cases, by SBA in four cases, and by HPA in one case. Since UEA I reactivity was characterized as dull and unclear, this lectin was excluded from Table 2. Since the binding of lectins in testicular tissue was influenced by the fixation method used (23), we compared the labelling results of the two testicular tumours (VP, MP) from which Bouinfixed tissue was available with those fixed in formalin. In general, the intensity of fluor-
~
Fig. 2. Histological sections of human embryonal carcinoma fixed in Bouin’s fluid. Phase contrast microphotographs of tumour (a) and (c), and corresponding areas stained with FITC-RCA 1 (b) and FITC-PNA (d). FITC-RCA I (b) labelled the cell borders and cytoplasm of carcinoma cells. The apical surfaces of cancer cells forming tubule-like structures showed particularly bright fluorescence (arrows). FITC-PNA (d) was positive in focal regions of carcinoma tissue. The labelling reaction was located along free borders of carcinoma cells (arrows), forming papillary structures. Magnification (a, b, c, d) x 425.
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escence was somewhat weaker in the formaldehyde-fixed than in the Bouin-fixed specimens, but slight changes in the staining pattern, depending on the fixative, were also observed. Parallel with paraffin-embedded material, acetonefixed frozen sections of the tumours were also used. Due to the poorly preserved morphology of such tumour cells, their staining results were not considered reliable. FITC-Con A was brightly bound to the cytoplasm of all the embryonal carcinoma cells; the fluorescence frequently appeared in a granular pattern (Figs. la, b). A similar heterogeneous distribution of granular reactivity among cancer cells was observed in tissues fixed in formalin or in Bouin. FITC-WGA labelled the cytoplasm of all tumour cells with an intense reaction along their luminal borders (Figs. lc, d) under both fixation conditions. In contrast, FITC-s-WGA showed weak staining of some tumour cells in the Bouinfixed specimens and was negative in the corresponding formalin-fixed tissues. FITC-RCA I demonstrated bright labelling of carcinoma cells, with a more pronounced staining on apical surfaces of the malignant cells (Figs. 2a, b). Some decrease in the intensity of the labelling reaction was observed in formalinfixed tissue samples. FITC-PNA was positive in the two Bouinfixed tumours. Fluorescence of some carcinoma cells was predominantly visualized on free cellular margins (Figs. 2c, d). The intensity of labelling was decreased in formalin-fixed tumours. FITC-SBA showed heterogeneous reactivity of some carcinoma cells in histological sections of the two Bouin-fixed tumours, while it was hardly positive in formalin-fixed tissues. FITCUEA I was also faintly positive when Bouin’s fluid was used for tissue fixation, but no clear staining was detected in formalin-fixed tissue.
FITC-HPA was negative in the carcinoma cells of the two tumours regardless of the fixative used. A few tubules with CIS germ cells were found in the periphery of Bouin-fixed (MP) and formalin-fixed (VT, HM, KH, SK, VP, MP) tissues of six testicular tumours. In all cases, Con A showed strong, often granular, reactivity in the cytoplasm of CIS cells. A less intense labelling of CIS germ cells was also achieved with WGA in all these tumours. RCA I was bound to all CIS cells in the Bouin-fixed tissue of the tumour (MP) and to some CIS cells in two formalinfixed tumours (VT, KH). S-WGA, PNA, SBA and HPA did not label CIS germ cells present adjacent to the Bouin- and formalin-fixed tumour tissue of these six embryonal carcinomas. The small seminomatous component of the tumour (VP) was strongly labelled with Con A. WGA and RCA I were also bound to the seminomatous cells of this tumour. Weak binding of s-WGA to some of the cells in the seminomatous tumour component was observed. PNA, SBA and HPA did not clearly label the seminoma cells of the combined tumour. Lectin binding properties of the stromal connective tissue and of the inflammatory cells infiltrating the tumours are not reported here as the main interest of the present study was to characterize the nature of embryonal carcinoma cells. In the control experiments, addition of inhibitory sugars to the lectin solutions resulted in either absence of or significant reduction in binding of the lectins. Xenograft tumour The xenograft tumour in nude mice, histologically representing embryonal carcinoma (Fig. 3a), showed a bright granular labelling of carcinoma cells by Con A (Fig. 3b). Malignant cells
Fig. 3. Histological sections of embryonal carcinoma implanted in nude mouse and stained with haematoxylin and eosin (a), FITC-Con A (b), FITC-WGA (c) and FITC-RCA I (d). (a) Embryonal carcinoma cells with large atypical nuclei (arrows) showed abundant mitotic figures. The general pattern of embryonal carcinoma was preserved in the transplant. FITC-Con A (b) showed a cytoplasmic labelling reaction which was granular in the majority of the malignant cells (arrows). FITC-WGA (c) was evenly bound to the cytoplasm of carcinoma cells (arrows), but labelling was more intense on free borders of cancer cells. FITC-RCA I (d) labelled cell borders brightly with a more prominent fluorescence along luminal margins of tubular structures (arrows). Magnification (a, b) x 350; (c, d) x 425.
238
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were also labelled by WGA and RCA I with an intense fluorescence along apical borders of tumour cells (Figs. 3c, d), forming small tubular structures. Some faint reactivity was observed by s-WGA. The tumour cells were stained by PNA, displaying a linear fluorescence of free cellular margins. For SBA and UEA I, dull and faint fluorescence of the carcinoma was seen. HPA did not label the murine carcinoma cells at all. Embryonul carcinoma cells in vitro Three cell lines of human embryonal carcinoma H 12.1, H 23.1.2, and 1428 A displayed typical morphological characteristics of embryonal carcinoma cells (Fig. 4a). Various cell lines were stained by Con A, which presented cytoplasmic fluorescence that, in some of the cells, was granular (Fig. 4b). The positive labelling reaction by WGA in all three cell lines was located especially along cell surfaces and in the perinuclear region of the cultured cells (Fig. 4c). S-WGA was weakly bound to the cytoplasm of carcinoma cells in all three cell lines. The cell lines were also stained by RCA I with a labelling reaction in the cytoplasm and along cell borders of embryonal carcinoma cells. PNA was faintly positive, whereas SBA did not clearly label malignant cells. Various cell lines failed to react with HPA. On the other hand, some dull intracellular labelling was noted by UEA I. DISCUSSION Using a panel of lectins we found histochemically that embryonal carcinoma cells from both patients with testicular cancer as well as experimental models were positively labelled by Con A, WGA, and RCA I. Other lectins showed focal and less uniform binding patterns of the malignant cells. In tumour cells, the presence of a great number of mannose- and glucose-containing glycoconjugates is demonstrated by positive cytoplasmic reactivity with Con A, as the lectin binds to polymannosyl cores of carbohydrate chains (20). The intensely granular Con A labelling in a portion of the malignant cells of each tumour suggests the heterogeneous nature of the tumour cell population. The accumulation of carbohydrate-rich substances in the cytoplasm may 240
Fig. 4. Embryonal carcinoma cells grown in a monolayer (H l2.l), fixed in 3.5% paraformaldehyde. Phase contrast microphotograph (a) and cells stained with FITC-Con A (b) and FITC-WGA (c). FITCCon A (b) was bound in a granular way to the cytoplasm of most of the tumour cells (arrows), whereas FITC-WGA (c) showed a more uniform labelling of cultivated cells (arrows). Magnification (a, b) x 425; (c) x 270.
partially be explained by defective membrane incorporation or secretion of glycoconjugates, as reported for other malignancies (1 7). Further explanations may also include the accelerated
LECTINS IN TESTICULAR CANCER
production of glycosylated proteins, such as alpha-fetoprotein and human chorionic gonadotrophin, which are the most important tumour markers of non-seminomatous germ cell tumours and are known to interact with Con A (15, 16). In addition to the less intense cytoplasmic staining, WGA binding sites were predominantly located on the luminal surfaces of carcinoma cells, forming tubular structures. This indicates a polarization of the embryonal carcinoma cells which was lost during cell culture, and which thus may be due to stromal factors having an influence on cell polarity. Similar polarity has also been found in mammary carcinoma with the apocrine membrane antigen, which sometimes also seems to persist in cell culture (10). WGA specifically binds sialic acid and N-acetylglucosamine (2, 12). In contrast, its succinylated derivative, s-WGA, is specific for N-acetylglucosamine only (25). As s-WGA was faintly positive or in many cases negative in different tumours, the bright fluorescence by WGA was apparently due to the high content of sialic acid in embryonal carcinoma cells. A similar increase in the sialic acid content is reported for many other carcinomas e.g. in breast (31), kidney (9), and colon (14). Lectin binding polarity of the tumour cells was also demonstrated by RCA I, suggesting the presence of terminal galactose and galactose residues at the penultimate position when substituted by sialic acids (8). Another galactose-specific lectin PNA, with a more limited binding specificity (12), focally labelled part of the embryonal carcinoma cells in both the Bouin-fixed tumours and in 10 formalin-fixed testicular tumours. The heterogeneous labelling of embryonal carcinoma cells observed with PNA apparently reflects either functionally or differentially related characteristics of individual cells, substantiating the results reported by other investigators (28, 32). On the other hand, PNA was more sensitive to the fixation method used in this study than were Con A, WGA, s-WGA, and RCA I. The lectin staining is clearly decreased when formaldehyde is used as fixative (23). Embryonal carcinoma cells showed heterogeneous reactivity to SBA, HPA and UEA I which was not dependent on the possible combination of other histological types with embry-
onal carcinoma. SBA recognizes N-acetylgalactosamine residues (12). Only four tumours presented some positive foci with this lectin. Another N-acetylgalactosamine-specific (1 2) lectin HPA was negative in almost all cases; one formalin-fixed tumour (JK), derived from undescended testis and with an aggressive clinical behaviour, reacted positively to HPA. The functional significance of this observation remains speculative, as a systematic correlation of lectin binding with the clinical data of the testicular cancer patients was not done during the study. The fucose-specific UEA I presented an unclear and dull reaction in histological sections of formalin-fixed tumours, yet it was positive in the Bouin-fixed specimens. Detection of the small amount of fucose found in some of the embryonal carcinomas was not verified only by the sensitivity to the fixative used (23), but is also reported by other investigators (32). The heterogeneous nature of embryonal carcinomas with respect to their sugar composition has also been shown by antibodies, e.g. by the anti-1(Ma) antibody which recognizes a branched carbohydrate chain composed of three N-acetyllactosamine units (32). Regardless of the germ cell origin, the expression of glucosyl moieties of embryonal carcinomas differed from that of normal germ cells ' during spermatogenic cell differentiation (2 1). When the lectin binding pattern of embryonal carcinomas is compared with that of testicular CIS and seminoma cells (21,22,24), the positive labelling of all varying cell types by Con A, WGA and RCA I suggests that the neoplastic cells are closely related. However, the specific differences in the distribution of binding sites by each lectin observed during this study and verified in the previous studies (21, 22, 32) illustrate that the nature of embryonal carcinomas is distinct from that of seminomas. The different features of embryonal carcinoma cells in comparison with seminoma cells are also reflected by PNA reactivity, as the majority of classic seminomas are negative for this lectin (19, 22, 24). In our study, even most of the formalinfixed tumours presented heterogeneous focal PNA reactivity, located on free cellular margins, adding evidence to the suggestion that the appearance of this marker is differentiation related (32). On the other hand, CIS germ cells and seminoma cells seem to have morphological fea24 1
MALMI & St)DERSTRt)M
tures (30) and patterns of glycosylation (22) in common. Therefore, the lectin histochemical results do not exclude the model of tumour pathogenesis in which embryonal carcinomas develop through a seminoma stage from intratubular CIS germ cells (7, 27). Otherwise, the regression of CIS germ cells into totipotent embryonal carcinoma cells (30) must be associated with distinct alterations in the distribution of cellular glycoconjugates. In the human germ cell tumour lines established to provide a model for the study of the differentiation of human germ cell tumours in vivo, the expression of Con A, WGA, RCA I and PNA binding sites was observed. The lectin staining pattern in histological sections of the xenograft tumour did not differ significantly from that of human embryonal carcinoma from testicular cancer patients. Our findings speak in favour of the usefulness of cell culture and animal models in studies of germ cell tumours. The results presented show that detection of cellular carbohydrates by lectin histochemistry is of potential significance in the investigation of the nature and origin of human embryonal carcinoma, both in histological sections and in experimental models. The use of lectins, in combination with other cellular markers and clinical data, provides an additional analytical tool for the study of human germ cell tumours. The skilful technical assistance of Mrs A . Vuoristo and Mr J. Liippo is kindly acknowledged. We thank Dr. C . Bokemeyer and Prof. H.-J. Schmoll for providing us with the human germ cell lines and the murine xenograft tumour. This study was supported by the Cancer Society and the Academy of Finland.
REFERENCES Ahoy, J., Ucci, A . A . & Pereira, M . E.: Lectin Histochemistry: An update. In: DeLellis, R . A . (Ed.): Advances in Immunohistochemistry, Raven Press, New York, 93-131, 1988. Bhavanandan, K & Katlic, A.: The interaction of wheat germ agglutinin with sialoglycoproteins the role of sialic acid. J. Biol. Chem. 254: 400W008, 1979. Casper, J., Schmoll. H.-J., Schnaidt, U. & Fonatsch, C.: Cell lines of human germinal cancer. Int. J. Androl. 10: 105-113, 1987. Casper, J . , Bronson, D. L., Harstrick, A . , Schnaidt, U. & Schmoll, H.-J.: Growth of human
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germ cell tumour cell lines in nude mice. In: Abstract book: Pathobiology of human germ cell neoplasia. Satellite Meeting of the 1 1 th International Congress of the International Society of Developmental Biologists, 1989 (abstr). 5. Damjanov, I.: Biology of disease. Lectin cytochemistry and histochemistry. Lab. Invest. 57: 5-20. 1987. 6. Damjanov, I.: Developmental biology of human germ cell tumours. In: Abstract book: Pathobiology of human germ cell neoplasia. Satellite Meeting of the 11th International Congress of the International Society of Developmental Biologists, 1989 (abstr). 7. Damjanov, I.: Is seminoma a relative or a precursor of embryonal carcinoma'? Lab. Invest. 60: 1-3, 1989. 8. Debray, H . . Decout, D., Strecker, G., Spik, G. & Montreul, J.: Specificity of twelve lectins towards oligosaccharides and glycopeptides related to Nglycosylproteins. Eur. J. Biochem. f f7: 41-55, 1981. 9. Echenique, J. E . & Graham S. D.: Significance of lipid-associated sialic acid and CA 19-9 as tumor markers for renal cell carcinoma. Urology 32: 397400, 1988. 10. Forsman, L.: An apocrine membrane antigen with polarized distribution and hormonally regulated expression in human endometrial and mammary carcinoma cell lines. APMIS 95: 315-323, 1987. 11. Giwercman, A , , Marks, A . , Bailey, D., Baumal, R. & Skakkehaek, N . E.: A monoclonal antibody as a marker for carcinoma in situ germ cells of the human adult testis. APMIS 96: 667-670, 1988. 12. Goldstein, I. J. & Hayes, C . E.: The lectins: Carbohydrate binding proteins of plants and animals. Adv. Carbohydr. Chem. Biochem. 35: 127-340, 1978. 13. Hustin, J., Collette, J . & Franchimont, I?: Immunohistochemical demonstration of placental alkaline phosphatase in various states of testicular development and in germ cell tumours. Int. J. Androl. 10: 29-35, 1987. 14. Irimura, i?, Carlson, D. A , , Price. J., Yumori, T , Giavazzi, R . , Ota, D. M . & Cleary, K . R.: Differential expression of a sialoglycoprotein with an approximate molecular weight of 900,000 on metastatic human colon carcinoma cells growing in culture and in tumor tissues. Cancer Res. 48: 2353-2360, 1988. 15. Ishiguro, T , Sawada, M . , Sakaguchi, H . & Sugitachi, I.: Alpha-fetoprotein subfractions in germ cell tumours identified by Con A or LCH crossedline affinity immunoelectrophoresis. AJRI 5: 102-105, 1984. 16. Koyama, K., Toda, K . , Kuriyama, D. & Isojima, S.: Affinity to lectin, biological and immunological characteristics of human chorionic gonado-
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17.
18.
19.
20.
tropins from pregnant women and trophoblastic tumour patients. Acta Endocrinol. 112: 579-585, 1986. Langkilde, N . C., WolJ H . & Orntoft, T E: Lectinhistochemistry of human bladder cancer: Loss of lectin binding structures in invasive carcinomas. APMIS 97: 367-373, 1989. Leathern, A.: Lectin histochemistry. In: Polak, J. M . , van Noorden, S. (Eds): Immunocytochemistry. Modern Methods and Applications. Second Edition. 167-187, 1986. Lee, M.-C., Talerman, A . , Oosterhuis, J. W & Damjanov, I.; Lectin histochemistry of classic and spermatocytic seminoma. Arch. Pathol. Lab. Med. 109: 938-942, 1985. Lotan. R . & Nicholson. G.: Purification of cell membrane glycoproteins by lectin affinity chromatography. Biochem. Biophys. Acta 559: 7 7 0 77c, 1n-m
J L 7 - J / U I 1717.
21. Malmi, R. & Soderstrom, K.-0.: Lectin binding sites in human seminiferous epithelium, in CIS cells and in seminoma. Int. J. Androl. 10: 157-162, 1987. 22. Malmi, R . & Soderstrom, K.-0.: Lectin binding to carcinoma-in-situ cells of the testis. A comparative study of CIS germ cells and seminoma Virchows Arch. A Anat) 413: 69-75, 1988. 23. Malmi, R. & Soderstrom, K.-0.: Lectin binding to rat spermatogenic cells: effects of different fixation methods and proteolytic enzyme treatment. Histochem. J. 20; 276282, 1988. 24. Malmi, R.. Kellokumpu-Lehtinen, I?-L. & Siiderstrom, K.-0.: Correlation between lectin binding and clinical factors in seminoma patients. J. Cancer Res. Clin. Oncol. 115: 96100, 1989.
25. Monsigny, M . . Sene, C . , Ohrenovitch, A , , Roche, A.-C., Delmotte, E & Boschetti, E: Properties of succinylated wheat germ agglutinin. Eur. J. Biochem. 98: 3945, 1979. 26. Mostofi, E K . & Sobin, L. H.: Histological typing of testis tumours. World Health Organization, Geneva, 1977. 27. Oosterhuis, J . W , Castedo, S. M . M . J . , de Jong, B., Cornelisse, C. J . , Dam, A . , Sleijyer, D. 7: & Schraffordt Koops, H.: Ploidy of primary germ cell tumours of the testis: pathogenetic and clinical relevance. Lab. Invest. 60: 14-21, 1989. 28. Reisner, Y , Gachelin, G . , Dubois, I?, Nicolas, J.-E, Sharon, N . & Jacob, F : Interaction of peanut agglutinin, a lectin specific for nonreducing terminal D-galactosyl residues, with embryonal carcinoma cells. Dev. Biol. 61: 2C27, 1977. 29. Skakkebaek, N . E.: Carcinoma-in-situ of the testis: frequency and relationship to invasive germ cell tumours in infertile men. Histopathol. 2: 157-170, 1978. 30. Skakkehaek, N . E . , Berthelsen, J . G . , Giwercman, A . & Miiller, J.: Carcinoma-in-situ of the testis: possible origin from gonocytes and precursor of all types of germ cell tumours except spermatocytoma. Int. J. Androl. 10: 19-28, 1987. 31. Stahli, C . , Caravatti, M . , Aeschbacher, M . , Kocyba, C.. Takacs, B. & Carmann, H.: Mucin-like carcinoma-associated antigen defined by three monoclonal antibodies against different epitopes. Cancer Res. 48: 6799-6802, 1988. 32. Teshima, s., Hirohashi, s., Shimosato, Z, Kishi, K . , Ino, Z, Matsumoto, K . & Yamada, T : Histochemically demonstrable changes in cell surface carbohydrates of human germ cell tumors. Lab. Invest. 50; 271-277, 1984.
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