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Germ cell tumors of the mediastinum and testis: A comparative immunohistochemical study of 120 cases
Germ Cell Tumors of the Mediastinum and Testis: A Comparative Immunohistochemical Study of 120 Cases SAUL SUSTER, MD, CESAR A, MORAN, MD, HUGO DOMINGUEZ-MALAGON, MD, AND PENELOPEQUEVEDO-BLANCO, BA An innnunohistochemical study was performed in 120 cases of mediastinal and testicular germ cell tumors from archival, paraffinembedded material to compare the patterns of expression between the two groups with a panel of markers, including broad-spectrum keratin, CAM 5.2 low-molecular-weight cytokerafin, placentul-like alkaline phosphatase (PLAP), alpha-fetoprotein (AFP), human [3-chorionic gonadotropin (hCG), vimentin, and CD30 (Ki-1 antigen). Significant differences were observed between mediastinal and testicular seminomas: mediastinal seminomas showed strong dot-like paranuclear positivity of the tumor cells with antibodies to CAM 5.2 low-molecular-weight keratin in 80% of cases (32 of 40), as compared with only 20% positivity (5 of 24) in testicular seminomas; placental alkaline phosphatase (PLAP) was also found to be less commonly expressed in testicular seminomas (12 of 24) than in mediasfinal seminomas (37 of 40); a similar pattern of expression was also observed for vimentin, which was present in scattered tumor cells in a higher proportion of mediastinal seminomas (28 of 40) than in testicular seminomas (11 of 24). The staining pattern and distribution of these markers did not show significant differences between the two
groups for the various other tumor categories studied, including yolk sac tumor, embryonal carcinoma, and choriocarcinoma. The tumor cells in both testicular and mediasfinal embryonal carcinoma showed strong positivity for the CD30 antigen; however, strong positivity for this marker was also observed in 6 of 25 yolk sac tumors and in scattered individual tumor cells in 4 of 63 seminomas. The results of this study show that significant differences exist between the inlmlmostaining patterns of mediastinal and testicular seminomas, suggesting that the former may be characterized by a more mature phenotype than their testlcular counterparts. Also, CD30 expression may not be necessarily restricted to embryonal carcinomas and occasionally may be observed in yolk sac tumors and seminoma cells, supporting the close histogenetic relationship that exists among these tumor types. HUM PATnOL 29:737-742. Copyright © 1998 by W.B. Satmders Company Key words: germ cell tumors, mediastinmn, immunohistochemistry, CD30, seminoma. Abbreviations: PLAP, placental-like alkaline phosphatase; AFP, alpha-fetoprotein; hCG, human chorionic gonadotropin.
E x t r a g o n a d a l g e r m cell t u m o r s a r e r a r e n e o p l a s m s t h a t h i s t o l o g i c a l l y closely r e s e m b l e t h e i r g o n a d a l c o u n terparts. Although the immunohistochemical staining p a t t e r n o f g e r m cell t u m o r s a r i s i n g in t h e g o n a d s has b e e n well s t u d i e d in t h e past, 1-s few if a n y c o m p r e h e n sive studies have b e e n p e r f o r m e d in these t u m o r s in e x t r a g o n a d a l locations. M o r e o v e r , a c o m p a r a t i v e s t u d y to d e t e r m i n e w h e t h e r s i g n i f i c a n t i m m u n o h i s t o c h e m i cal d i f f e r e n c e s exist b e t w e e n t h e g o n a d a l a n d e x t r a g o n a d a l lesions has b e e n lacking. W e have s t u d i e d a series o f 120 archival cases o f g o n a d a l a n d e x t r a g o n a d a l g e r m cell t u m o r s with a p a n e l o f i m m u n o h i s t o c h e m i c a l cell m a r k e r s o n f o r m a l i n fixed, p a r a f f i n - e m b e d d e d m a t e r i a l . T h e results o f o u r s t u d y i n d i c a t e t h a t s i g n i f i c a n t d i f f e r e n c e s m a y exist b e t w e e n t e s t i c u l a r a n d m e d i a s t i n a l lesions, p a r t i c u l a r l y among seminomas. The implications of these findings for the pathogenesis and diagnosis of these tumors are discussed.
MATERIALS AND METHODS
From the Arkadi M. Rywlin Department of Pathology & Laboratory Medicine, Mount Sinai Medical Center, and University of Miami School of Medicine, Miami, FL; the Department of Pulmonary & Mediastinal Pathology, Armed Forces Institute of Pathology, Washington, DC; and the National Cancer Institute, Mexico City, Mexico. Accepted for publication November 14, 1997. Address correspondence and reprint requests to Saul Suster, MD, Department of Pathology, Mount Sinai Medical Center, 4300 Alton Rd, Miami Beach, FL 33140. Copyright © 1998 by W.B. Saunders Company
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One h u n d r e d twenty cases of primary mediastinal and testicular germ cell tumors were retrieved from the files of the Department of Pathology and Laboratory Medicine, Mount Sinai Medical Center of Greater Miami, Florida (38 cases), the Department of Pulmonary and Mediastinal Pathology, Armed Forces Institute of Pathology, Washington, DC (52 cases), and the Department of Pathology, National Cancer Institute, Mexico City, Mexico (30 cases). The tumors included 24 testicular and 40 mediastinal seminomas, seven testicular and 18 mediastinal yolk sac tumors, 19 testicular and three mediastinal embryonal carcinomas, and three testicular and six mediastinal choriocarcinomas. All patients were adult men ranging in age from 21 to 79 years (mean, 42). All the tumors were categorized according to the World Health Organization criteria. 9 Representative paraffin blocks from formalin-fixed tissue were available in all cases for immunohistochemical studies. Tissue sections from the blocks selected for immunohistochemical study were first screened to ensure that the tissue examined contained elements that were representative histologically of tile different tumor categories studied, and contained only pure forms of the various tumor types. Tissue sections were incubated with antibodies against broad-spectrum keratin (Dako, Carpinteria, CA; 1:3000), CAM 5,2 cytokeratin (Beckton-Dickinson, San Jose, CA), vimentin (Dako, clone V9), placental-like alkaline phosphatase (PLAP) (Dako; 1:200), c~-fetoprotein (AFP) (Dako; 1:3), human [3-chorionic gonadotropin (hCG) (Dako), and CD30 (Ber-H2 clone) (Dako; 1:30) by the avidin-biotin-peroxidase complex technique. The sections incubated with antibodies to broad-spectrum keratin, CAM 5.2 cytokeratin, vimentin, AFP, PLAP, and CD30 were treated using a microwave epitope
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retrieval technique with 10 mmol/L citrate buffer, pH 6.0, at high temperature for 20 minutes. Appropriate positive controls were run concurrently for all the antibodies tested. Nonimmune mouse and rabbit serum was substituted for negative controls. Results of the immunohistochemical reaction were semiquantitated according to the following scale: (1) negative = no staining observed in the tumor cells; (2) focally positive = less than 25% of tumor cells were positive; (3) positive = 25% to 75% of tumor cells positive; and (4) strongly positive = greater than 75% of tumor cells positive.
testicular s e m i n o m a and in one case of mediastinal s e m i n o m a with the CD30 antigen (Fig 3). Yolk Sac Tumor All cases of mediastinal (18 of 18) and testicular (seven of seven) yolk sac t u m o r were strongly positive for CAM 5.2 antibodies (Fig 4). N o n e of the cases i m m u n o r e a c t e d with broad-spectrum keratin antibodies. AFP was m o r e often expressed in testicular yolk sac t u m o r (six of seven) than in mediastinal tumors (10 of 18). Stains for PLAP and h C G were uniformly negative in all cases. Stains for vimentin showed focal, weak cytoplasmic positivity in scattered t u m o r cells in four of seven testicular tumors and 6 of 18 mediastinal tumors. An interesting finding was the presence of focal m e m b r a n e and cytoplasmic positivity of the t u m o r cells in four of seven cases of testicular yolk sac t u m o r and 2 of 18 mediastinal cases with antibodies for CD30 (Fig 5).
RESULTS T h e results of i m m u n o h i s t o c h e m i c a l stains are summarized in Table 1. Seminomas T h e r e was a notable difference between the staining pattern of mediastinal a n d testicular seminomas for certain markers in the study: mediastinal seminomas showed a striking dot-like paranuclear positivity with CAM 5.2 low-molecular-weight cytokeratin in 32 of 40 as c o m p a r e d with 5 of 24 in testicular seminomas (Fig 1). Strong dot-like paranuclear positivity for CAM 5.2 was also observed in cells within n o r m a l seminiferous tubules a n d in foci of intratubular (in situ) g e r m cell neoplasia adjacent to testicular seminomas in all 12 cases in which these elements were present in the sections available for study. Stains for broad-spectrum keratin also showed focal cytoplasmic positivity o f t u m o r cells in 27 of 40 mediastinal seminomas; this staining, however, was less intense and was seen in m u c h fewer cells than with CAM 5.2. N o n e of the testicular seminomas in the study showed positivity for broad-spectrum keratin (0 of 24). Vimentin showed focal cytoplasmic positivity in single scattered cells of mediastinal seminomas in 28 of 40 cases, and in 11 of 24 cases of testicular seminomas; this stain a p p e a r e d to be m o r e intense and present in a larger n u m b e r of cells in the mediastinal cases (Fig 2). PLAP was also m o r e c o m m o n l y f o u n d in mediastinal seminomas (37 of 40) than in testicular seminomas (12 of 24). H C G positivity was f o u n d in isolated syncytiotrophoblastic-type multinucleated tum o r cells in 4 o f 24 testicular seminomas and in one mediastinal seminoma. Stains ibr AFP were uniformly negative in all cases studied. Interestingly, focal m e m b r a n e and cytoplasmic positivity could be observed in isolated, scattered single cells in 3 of 24 cases of TABLE 1,
CAM5.2 Keratin PLAP AFP hCG VIM CD30
All cases of e m b r y o n a l carcinoma were strongly positive for CAM 5.2 low-molecular-weight cytokeratin (Fig 6). In contrast, only 2 o f 19 cases o f testicular e m b r y o n a l carcinoma and n o n e of the mediastinal cases showed positivity for broad-spectrum keratin. T h e r e was focal positivity observed in small clusters or in isolated t u m o r cells in 6 of 19 cases of e m b r y o n a l carcinoma with AFP, and in 5 of 19 cases with PLAP. Stains for h C G showed focal positivity in 5 of 19 testicular tumors and in o n e of three mediastinal cases. Vimentin showed focal positivity in scattered, isolated t u m o r cells in 3 of 19 testicular tumors, and was negative in all three mediastinal lesions. CD30 showed strong positivity in 14 of 19 testicular tumors; this stain showed a m e m b r a n e and focal paranuclear cytoplasmic reaction in 12 cases, and diffuse cytoplasmic reaction in two (Fig 7). Focal CD30 positivity was also observed in o n e case of mediastinal e m b r y o n a l carcinoma; the staining reaction was both cytoplasmic (diffuse) and m e m b r a nous. Choriocarcinoma All cases of testicular and mediastinal choriocarcin o m a showed diffuse cytoplasmic positivity of the t u m o r cells with CAM 5.2 a n d broad-spectrum keratin antibodies. T h e intensity of the staining reaction was stronger
Resultsof Immunohistochemical Stains in 120 Cases of Germ Cell Tumors of the Mediastinum and Testis Seminoma
Stain
Embryonal C a r c i n o m a
Mediastinum 32/40 27/40 37/40 0 1/40 28/40 1/40
Yolk Sac Tumor Testis
Mediastinum
5/24 0/24 12/24 0 4/24 11/24 3/24
18/18 0 0 10/18 0 6/18 2/18
Embryonal Carcinoma
Testis 7/7 0 0 6/7 0 4/7 4/7
Mediastinum 3/3 0 0 0 1/3 0 1/3
Testis 19/19 2/19 5/19 6/19 5/19 3/19 14/19
Choriocarcinoma Mediastinum 6/6 6/6 0 0 6/6 0 0
Testis 3/3 3/3 0 0 3/3 0 0
Abbreviations: PLAIt,placental-like alkaline phosphatase; AFP, alpha-fetoprotein; hCG, human [3-chorionicgonadotropin; VIM,vimentin.
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MEDIASTINAL AND TESTICULARGERM CELL TUMORS (Suster et al)
!:
FIGURE 1. Mediastinal seminoma showing paranuclear dot-like CAM 5.2 low-molecular-weight cytokeratin positivity.
FIGURE 3. Strong membrane positivity is seen in this seminoma cell for the CD30 antigen.
The immunohistochemical profile of germ cell tumors in gonadal locations has been extensively studied in the past, often with conflicting results being reported in the literature. 8,~°q5 The immunostaining pattern of extragonadal germ cell tumors, howevm, has not been as extensively investigated. In particular, no comprehensive studies in large series of cases have been available to compare the immunohistochemical profile of the gonadal tumors with those arising in extragonadal locations. We examined 67 mediastinal and 53 testicular germ cell tumors using a panel o f immunohistochemical stains in sections taken from archival, paraffin-
e m b e d d e d material. The most striking differences were f o u n d in the group of seminomas: mediastinal seminomas were characterized by a striking dot-like paranuclear positivity with CAM 5.9 antibodies in 32 of 40 cases, compared with testicular seminomas in which a similar staining pattern was observed in only 5 of 24 cases. Mediastinal seminomas also showed focal cytoplasmic positivity with broad-spectrum keratin antibodies in 97 of 40 cases, whereas testicular seminomas were uniformly negative for this antibody. Stains for PLAP and vimentin were also f o u n d to be more frequently positive in mediastinal than in testicular seminomas. Another interesting finding was the demonstration of focal positivity with CD30 antibodies in isolated t u m o r cells in four cases of seminoma, and in clusters of tumors cells in six cases of yolk sac tumor. No significant differences between the staining patterns of testicular and mediastinal tumors could be identified for any of the other tumor categories in the study, including yolk sac tumor, embryonal carcinoma, and choriocarcinoma. The differences of expression for cytokeratin intermediate filaments between mediastinal and testicular seminomas is of interest and may have some bearing on recent observations regarding the pathogenesis of these lesions. Results of molecular, cytogenetic, and DNA
FIGURE 2. positivity.
FIGURE 4. Yolk sac tumor of the mediastinum showing strong CAM 5,2 low-molecular-weight cytokeratin positivity.
with CAM 5.2 than with broad-spectrum keratin. T h e strongest staining reaction was observed in the cytotrophoblastic components, although syncytiotrophoblastic cells were also positive. All cases in the study also showed strong positivity of the syncytiotrophoblastic c o m p o n e n t for hCG. Stains for AFP, PLAP, vimentin, and CD30 were negative in the tumor cells of all cases tested.
DISCUSSION
Mediastinal seminoma showing focal vimentin
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4 '-t
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FIGURE 5. Yolksac tumor showing membrane positivity of the tumor cells for CD30.
FIGURE 7. Embryonal carcinoma showing strong membrane and focal dot-like paranuclear positMty for CD30.
ploidy studies appear to indicate that significant differences may exist between mediastinal and testicular seminomas. In a flow cytometric study of mediastinal germ cell tumors, Oosterhuis et a116 were able to demonstrate that most cases o f s e m i n o m a in the mediastinum were diploid, in comparison with testicular seminomas of the adult, which are predominantly aneuploid. In another recent study comparing k-ras-2 gene sequence analysis and expression of p53 between mediastinal and testicular seminomas, we were able to demonstrate the presence of a single-codon 13 k-ras point mutation in 1 of 13 mediastinal seminomas, while the rest of the tumors showed a normal germline configuration. 17 This stands in contrast with most of the previously published studies, which have reported an incidence as high as 40% of K-ras codon-12 point mutations in testicular seminomas. 18,19Additionally, we were able to show that p53 was only weakly and focally expressed in 4 of 13 cases of mediastinal seminoma (31%), as compared with 10 of 13 cases of testicular seminoma (77%). The expression of keratin and vimentin intermediate filaments similarly appears to point to differences between testicular and mediastinal seminomas. Some of
FIGURE6. Embryonal carcinoma showing strong CAM 5.2 low-molecuLar-weight cytokeratin positivity. 740
the early immunohistochemical studies reported in the literature appeared to indicate that seminoma ceils did not contain cytokeratin intermediate filaments. ~°,a2,2°In a study of testicular seminomas using affinity-purified antibodies by indirect immunofluorescence by Denk et al, 13 the authors f o u n d that most of the tumor cells appeared to lack cytokeratin-intermediate filaments, yet most of the cells contained desmosomes, as demonstrated by strong labeling with desmoplakin antibodies, indicative of early or incipient epithelial differentiation. It was further n o t e d that many of the seminoma ceils contained sparse vimentin filaments and that the arr a n g e m e n t and density o f these cytoskeletal intermediate filaments varied with the degree of maturation of the tissue. T h e most regular distribution and intensive staining of cytokeratin and desmoplakin was observed in the more " m a t u r e " tissues. A subsequent study by Niehans et al s in a large series of cases of germ cell tumors using a "cocktail" o f monoclonal cytokeratin antibodies (including CAM 5.2, MAK-6, and AE1/AE3) using the avidin-biotin-peroxidase complex technique f o u n d focal to diffuse keratin positivity in up to 10% of seminomas. Another study by Fogel et a115again demonstrated the heterogeneity of intermediate filament expression in testicular seminoma, with some of the cases showing neither cytokeratin n o r vimentin, some of the cases showing only cytokeratin or vimentin, and more than half of the cases showing co-expression of keratin and vimentin intermediate filaments. Whereas in most of the cases in that study, the cytokeratin subtypes present were mostly of the "simple epithelia" type (CK 8 and 18), in two cases the seminoma ceils also contained cytokeratins 4 and 12, which are normally f o u n d in stratified or complex glandular epithelium, thus denoting a potential for a more advanced and specialized degree of epithelial differentiation. The finding of cytokeratin expression in up to 20% of cases of testicular seminoma in our study is thus in agreement with previous studies reported in the literature for these tumors. The finding of up to 80% o f keratin positivity in mediastinal seminomas in our study, however, requires further explanation. Traditionally, seminomas have been thought to correspond to terminally differentiated germ cell tu-
MEDIASTINAL AND TESTICULARGERM CELL TUMORS (Suster et al)
mors. 21 This view, however, has been challenged in recent years based on the observation of the frequent admixtures and transitions of seminoma with other types of germ cell tumors. 15,2z The demonstration of keratin, vimentin, and desmoplakin immunoreactivity in these tumors, which are markers that are not normally expressed in germ cells, ~3 lends additional support to the notion that seminomas may not represent terminally differentiated neoplasms but are capable of either somatic, embryonal, or trophoblastic differentiation u n d e r certain circumstances. Niehans et al 8 speculated that cytokeratin production by seminoma cells may be one of the earliest indications that such cells are committed toward embryonal, yolk sac, or trophoblastic differentiation, and that expression of these proteins may precede morphological changes detectable at the light microscopic level. The marked differences in expression of these markers between mediastinal and testicular seminoma observed in the current study could thus be attributed to the mediastinal tumors showing a higher degree of differentiation than the testicular neoplasms. This observation is in agreement with the results of DNA ploidy and cytogenetic studies, which all point toward a more stable, mature cellular population in mediastinal seminomas compared with the testicular tumors. 16,17The presence of a more stable, mature, and better differentiated tumor cell population in mediastinal seminoma, as demonstrated by the lower incidence of point mutations, their diploid cell population, low expression of p53, and higher content of cytokeratin and vimentin intermediate filaments, would also explain the better prognosis and lower incidence of metastasis and aggressive behavior of mediastinal semin o m a compared with their testicular counterparts. 24 T h e presence of CD30 positivity in occasional seminoma cells and yolk sac tumors in our study is also of interest. M t h o u g h originally described as an activation marker expressed in Hodgkin's disease and anaplastic large cell lymphoma, recent studies have also demonstrated expression of the CD30 antigen in embryonal carcinomas. ~527 In fact, it has been stated that CD30+ represents a distinctive feature of embryonal carcinoma that may be helpful for the differential diagnosis from other germ cell tumors. 25,27A more recent study, however, was able to detect immunohistochemical staining for CD30 in isolated seminoma cells in 4 of 21 cases of classic testicular seminoma. 2a Our findings confirm these observations and support the close histogenetic relationship that exists between seminoma and embryohal carcinoma. The finding of occasional isolated CD30+ seminoma cells can hardly be regarded as a serious pitfall for the diagnosis of these tumors and is merely of interest for its histogenetic implications. The finding of strong m e m b r a n e and cytoplasmic positivity for CD30 in yolk sac tumor, however, can potentially create serious difficulties for the diagnosis of these tumors. Awareness of this fact is therefore of importance in clinical practice to avoid misinterpreting CD30 positivity in an otherwise typical yolk sac t u m o r for evidence of embryonal carcinomatous differentiation. Such cases should be evaluated within the context of the
morphology and with the aid of an additional panel of immunostains for adequate interpretation. Along similar lines, the strong dot-like cytokeratin positivity observed in mediastinal seminomas, although of limited diagnostic value, may introduce difficulties for diagnosis, particularly in small mediastinoscopic biopsy specimens, in which the cytokeratin positivity could be misinterpreted as indicating the presence of a carcinoma, either primary or metastatic to the thymus. Attention to the distinctive dot-like paranuclear pattern of staining, however, should alert the pathologist to the possibility that she or he is dealing with a mediastinal seminoma, and should p r o m p t the search for immunoreactivity with other more specific antibodies. In summary, the current study appears to indicate the existence of significant differences in immunostaining pattern between mediastinal and testicular seminomas, in particular for cytokeratin and vimentin intermediate filaments. These differences may point toward a more mature cell population with a more advanced stage of differentiation for the tumor cells in mediastinal seminomas. Additionally, the identification of CD30 immunoreactivity in seminoma and yolk sac tumor, in addition to embryonal carcinoma, appears to further highlight the close histogenetic relationship that exists among these tumors.
REFERENCES 1. Palmer PE, Safaii H, Wolfe HJ: Alpha-l-antitrypsin and alphafetoprotein: Protein markers in endodermal sinus (yolk sac) tumors. A m J Clin Pathol 65:575-582, 1976 2. Kurman RJ, Scardino PT, McIntire KR, et ah Cellular localization of alpha-fetoprotein and human chorionic gonadotropin in germ cell tumors of the testis using an indirect immunoperoxidase technique: A new approach to classification utilizing tumor markers. Cancer 40:2136-2151, 1977 3. BeilbyJOW, H o m e CHW, Milne GD, et al: Alpha-fetoprotein, alpha-l-antitrypsin, and transferrin in gonadal yolk sac tumors.J Clin Patho132:455-461, 1979 4. Jacobsen GK, Norgaard-Pedersen B: Placental alkaline phosphatase in testicular germ cell tumors and in carcinoma-in-situ of the testis: An immunohistochemical study. Acta Pathol Microbiol Immunol Scand [A] 92:323-329, 1984 5. Uchida T, Shimoda T, Miyata H, et al: Immunoperoxidase study of alkaline phosphatase in testicular tumors. Cancer 48:14551462, 1985 6. Manivel JC, Jessurun J, Wick MR, et al: Placental alkaline phosphatase immunoreactivity in testicular germ cell neoplasms. Am J Surg Pathol 11:21-29, 1987 7. Jacobsen GK, Jacobsen M, Clausen PP: Distribution of tumorassociated antigens in the various histologic components of germ cell tumors of the testis. AnlJ Surg Pathol 5:257-266, 1981 8. Niehans GA, ManiveIJC, Copland GT, et al: Immunohistochemistry of germ cell and trophoblastic neoplasms. Cancer 62:1113-1123, 1988 9. Mostofi ~T,, Sobin LH: Histological typing of testicular tumors, in International Histological Classification of Tumors, vol 16. Geneva, Switzerland, World Health Organization, 1977 10. Battifora H, Sheibani K, Tubbs RR, et ah Antikeratin antibodies in tumor diagnosis: Distinction between seminoma and embryonal carcinoma. Cancer 54:843-848, 1984 11. Miettinen M, Virtanen I, Talerman A: Intermediate filament proteins in human testis and testicular germ cell tumors. Am J Pathol 120:402-410, 1985 12. Raemaekers F, Feitz W, Moesker O, et ah Antibodies to cytokeratin and ~dmentin in testicular tumor diagnosis. Virchows Arch [A] 408:12%142, 1985
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13. Denk H, Moll R, Weybora W, et al: Intermediate filaments and desmosomal plaque proteins in testicular seminoma and nonseminomatous germ cell tumors as revealed by immunohistochemistry. Virchows Arch [A] 410:295-307, 1987 14. BenfleyAJ, Parkinson MC, Harding BN, et al: A comparative morphological and immunohistochemical study of testicular seminoma and intracranial germinoma. Histopathology 17:443-449, 1990 15. Fogel M, Lifschitz-Mercer B, Moll R, et al: Heterogeneity of intermediate filament expression in human testicular seminomas. Differentiation 45:242-249, 1990 16. OosterhuisJW, Rammeloo RHU, Cornelisse CJ, et al: Ploidy of malignant mediastinal germ cell tumors. HuM PATHOL 21:729-732, 1990 17. Przygodzki RM, Moran CA, Suster S, et al: Primary mediastinal and testicular seminomas: A comparison of K-ras-2 gene sequence and p53 immunoperoxidase analysis of 25 cases. HuM PATHOL27:975979, 1996 18. MoulJW, Theune SM, Chang EH: Detection ofras mutations in archival testicular germ cell tumors by polymerase chain reaction and oligonucleotide hybridization. Genes Chromosom Cancer 5:109118, 1992 19. Mulder MR Keijzer W, Verbek A, et al: Activated ras oncogene in human seminoma: Evidence for tumor heterogeneity. Oncogene 4:1345-1351, 1989 20. NakagawaY, Perentes E, Ross GW, et al: Immunohistochemical differences between intracranial germinoma and their gonadal
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equivalents: An immunoperoxidase study of germ cell tumors with epithelial membrane antigen, cytokeratin, and vimentin. J Pathol 156:67-72, 1988 21. Teilum G: Classification of endodermal sinus tumor (mesothelioma vitellinum) and so-called "embryonal carcinoma" of the ovary. Acta Pathol Microbiol Scand 64:407-429, 1965 22. Skakkebaek NE, Berthelsen JG: Carcinoma in-situ of the testis and invasive growth of different types of germ cell tumors: A revised germ cell theory. IntJ Androl 4:26-34, 1981 23. Osborn M, Weber K: Tumor diagnosis by intermediate filament typing: A novel tool for surgical pathology. Lab Invest 48:372-394, 1983 24. Moran CA, Suster S, Przygodzki RM, et al: Germ cell tumors of the mediastinum. II. Mediastinal seminoma: A clinicopatholo~cal and immunohistochemical study of 120 eases. Cancer 80:691-698, 1997 25. Pallesen G, Hamilton-Dutoit ST: Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma. Am J Patho1133:446-450, I988 26. Latza U, Foss H-D, Durkop H, et al: CD30 antigen in embryonal carcinoma and embryogenesis and release of the soluble molecule. AnlJ Patho1146:463-471, 1995 27. Ferreiro JA: Ber-H2 expression in tesficular germ cell tumors. HUM PATHOL25:522-524, 1994 28. Hittmair A, Rogatsch H, Hobisch A, et al: CD30 expression in seminoma. HUN PATttOL27:1166-1171, 1996
groups for the various other tumor categories studied, including yolk sac tumor, embryonal carcinoma, and choriocarcinoma. The tumor cells in both testicular and mediasfinal embryonal carcinoma showed strong positivity for the CD30 antigen; however, strong positivity for this marker was also observed in 6 of 25 yolk sac tumors and in scattered individual tumor cells in 4 of 63 seminomas. The results of this study show that significant differences exist between the inlmlmostaining patterns of mediastinal and testicular seminomas, suggesting that the former may be characterized by a more mature phenotype than their testlcular counterparts. Also, CD30 expression may not be necessarily restricted to embryonal carcinomas and occasionally may be observed in yolk sac tumors and seminoma cells, supporting the close histogenetic relationship that exists among these tumor types. HUM PATnOL 29:737-742. Copyright © 1998 by W.B. Satmders Company Key words: germ cell tumors, mediastinmn, immunohistochemistry, CD30, seminoma. Abbreviations: PLAP, placental-like alkaline phosphatase; AFP, alpha-fetoprotein; hCG, human chorionic gonadotropin.
E x t r a g o n a d a l g e r m cell t u m o r s a r e r a r e n e o p l a s m s t h a t h i s t o l o g i c a l l y closely r e s e m b l e t h e i r g o n a d a l c o u n terparts. Although the immunohistochemical staining p a t t e r n o f g e r m cell t u m o r s a r i s i n g in t h e g o n a d s has b e e n well s t u d i e d in t h e past, 1-s few if a n y c o m p r e h e n sive studies have b e e n p e r f o r m e d in these t u m o r s in e x t r a g o n a d a l locations. M o r e o v e r , a c o m p a r a t i v e s t u d y to d e t e r m i n e w h e t h e r s i g n i f i c a n t i m m u n o h i s t o c h e m i cal d i f f e r e n c e s exist b e t w e e n t h e g o n a d a l a n d e x t r a g o n a d a l lesions has b e e n lacking. W e have s t u d i e d a series o f 120 archival cases o f g o n a d a l a n d e x t r a g o n a d a l g e r m cell t u m o r s with a p a n e l o f i m m u n o h i s t o c h e m i c a l cell m a r k e r s o n f o r m a l i n fixed, p a r a f f i n - e m b e d d e d m a t e r i a l . T h e results o f o u r s t u d y i n d i c a t e t h a t s i g n i f i c a n t d i f f e r e n c e s m a y exist b e t w e e n t e s t i c u l a r a n d m e d i a s t i n a l lesions, p a r t i c u l a r l y among seminomas. The implications of these findings for the pathogenesis and diagnosis of these tumors are discussed.
MATERIALS AND METHODS
From the Arkadi M. Rywlin Department of Pathology & Laboratory Medicine, Mount Sinai Medical Center, and University of Miami School of Medicine, Miami, FL; the Department of Pulmonary & Mediastinal Pathology, Armed Forces Institute of Pathology, Washington, DC; and the National Cancer Institute, Mexico City, Mexico. Accepted for publication November 14, 1997. Address correspondence and reprint requests to Saul Suster, MD, Department of Pathology, Mount Sinai Medical Center, 4300 Alton Rd, Miami Beach, FL 33140. Copyright © 1998 by W.B. Saunders Company
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One h u n d r e d twenty cases of primary mediastinal and testicular germ cell tumors were retrieved from the files of the Department of Pathology and Laboratory Medicine, Mount Sinai Medical Center of Greater Miami, Florida (38 cases), the Department of Pulmonary and Mediastinal Pathology, Armed Forces Institute of Pathology, Washington, DC (52 cases), and the Department of Pathology, National Cancer Institute, Mexico City, Mexico (30 cases). The tumors included 24 testicular and 40 mediastinal seminomas, seven testicular and 18 mediastinal yolk sac tumors, 19 testicular and three mediastinal embryonal carcinomas, and three testicular and six mediastinal choriocarcinomas. All patients were adult men ranging in age from 21 to 79 years (mean, 42). All the tumors were categorized according to the World Health Organization criteria. 9 Representative paraffin blocks from formalin-fixed tissue were available in all cases for immunohistochemical studies. Tissue sections from the blocks selected for immunohistochemical study were first screened to ensure that the tissue examined contained elements that were representative histologically of tile different tumor categories studied, and contained only pure forms of the various tumor types. Tissue sections were incubated with antibodies against broad-spectrum keratin (Dako, Carpinteria, CA; 1:3000), CAM 5,2 cytokeratin (Beckton-Dickinson, San Jose, CA), vimentin (Dako, clone V9), placental-like alkaline phosphatase (PLAP) (Dako; 1:200), c~-fetoprotein (AFP) (Dako; 1:3), human [3-chorionic gonadotropin (hCG) (Dako), and CD30 (Ber-H2 clone) (Dako; 1:30) by the avidin-biotin-peroxidase complex technique. The sections incubated with antibodies to broad-spectrum keratin, CAM 5.2 cytokeratin, vimentin, AFP, PLAP, and CD30 were treated using a microwave epitope
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retrieval technique with 10 mmol/L citrate buffer, pH 6.0, at high temperature for 20 minutes. Appropriate positive controls were run concurrently for all the antibodies tested. Nonimmune mouse and rabbit serum was substituted for negative controls. Results of the immunohistochemical reaction were semiquantitated according to the following scale: (1) negative = no staining observed in the tumor cells; (2) focally positive = less than 25% of tumor cells were positive; (3) positive = 25% to 75% of tumor cells positive; and (4) strongly positive = greater than 75% of tumor cells positive.
testicular s e m i n o m a and in one case of mediastinal s e m i n o m a with the CD30 antigen (Fig 3). Yolk Sac Tumor All cases of mediastinal (18 of 18) and testicular (seven of seven) yolk sac t u m o r were strongly positive for CAM 5.2 antibodies (Fig 4). N o n e of the cases i m m u n o r e a c t e d with broad-spectrum keratin antibodies. AFP was m o r e often expressed in testicular yolk sac t u m o r (six of seven) than in mediastinal tumors (10 of 18). Stains for PLAP and h C G were uniformly negative in all cases. Stains for vimentin showed focal, weak cytoplasmic positivity in scattered t u m o r cells in four of seven testicular tumors and 6 of 18 mediastinal tumors. An interesting finding was the presence of focal m e m b r a n e and cytoplasmic positivity of the t u m o r cells in four of seven cases of testicular yolk sac t u m o r and 2 of 18 mediastinal cases with antibodies for CD30 (Fig 5).
RESULTS T h e results of i m m u n o h i s t o c h e m i c a l stains are summarized in Table 1. Seminomas T h e r e was a notable difference between the staining pattern of mediastinal a n d testicular seminomas for certain markers in the study: mediastinal seminomas showed a striking dot-like paranuclear positivity with CAM 5.2 low-molecular-weight cytokeratin in 32 of 40 as c o m p a r e d with 5 of 24 in testicular seminomas (Fig 1). Strong dot-like paranuclear positivity for CAM 5.2 was also observed in cells within n o r m a l seminiferous tubules a n d in foci of intratubular (in situ) g e r m cell neoplasia adjacent to testicular seminomas in all 12 cases in which these elements were present in the sections available for study. Stains for broad-spectrum keratin also showed focal cytoplasmic positivity o f t u m o r cells in 27 of 40 mediastinal seminomas; this staining, however, was less intense and was seen in m u c h fewer cells than with CAM 5.2. N o n e of the testicular seminomas in the study showed positivity for broad-spectrum keratin (0 of 24). Vimentin showed focal cytoplasmic positivity in single scattered cells of mediastinal seminomas in 28 of 40 cases, and in 11 of 24 cases of testicular seminomas; this stain a p p e a r e d to be m o r e intense and present in a larger n u m b e r of cells in the mediastinal cases (Fig 2). PLAP was also m o r e c o m m o n l y f o u n d in mediastinal seminomas (37 of 40) than in testicular seminomas (12 of 24). H C G positivity was f o u n d in isolated syncytiotrophoblastic-type multinucleated tum o r cells in 4 o f 24 testicular seminomas and in one mediastinal seminoma. Stains ibr AFP were uniformly negative in all cases studied. Interestingly, focal m e m b r a n e and cytoplasmic positivity could be observed in isolated, scattered single cells in 3 of 24 cases of TABLE 1,
CAM5.2 Keratin PLAP AFP hCG VIM CD30
All cases of e m b r y o n a l carcinoma were strongly positive for CAM 5.2 low-molecular-weight cytokeratin (Fig 6). In contrast, only 2 o f 19 cases o f testicular e m b r y o n a l carcinoma and n o n e of the mediastinal cases showed positivity for broad-spectrum keratin. T h e r e was focal positivity observed in small clusters or in isolated t u m o r cells in 6 of 19 cases of e m b r y o n a l carcinoma with AFP, and in 5 of 19 cases with PLAP. Stains for h C G showed focal positivity in 5 of 19 testicular tumors and in o n e of three mediastinal cases. Vimentin showed focal positivity in scattered, isolated t u m o r cells in 3 of 19 testicular tumors, and was negative in all three mediastinal lesions. CD30 showed strong positivity in 14 of 19 testicular tumors; this stain showed a m e m b r a n e and focal paranuclear cytoplasmic reaction in 12 cases, and diffuse cytoplasmic reaction in two (Fig 7). Focal CD30 positivity was also observed in o n e case of mediastinal e m b r y o n a l carcinoma; the staining reaction was both cytoplasmic (diffuse) and m e m b r a nous. Choriocarcinoma All cases of testicular and mediastinal choriocarcin o m a showed diffuse cytoplasmic positivity of the t u m o r cells with CAM 5.2 a n d broad-spectrum keratin antibodies. T h e intensity of the staining reaction was stronger
Resultsof Immunohistochemical Stains in 120 Cases of Germ Cell Tumors of the Mediastinum and Testis Seminoma
Stain
Embryonal C a r c i n o m a
Mediastinum 32/40 27/40 37/40 0 1/40 28/40 1/40
Yolk Sac Tumor Testis
Mediastinum
5/24 0/24 12/24 0 4/24 11/24 3/24
18/18 0 0 10/18 0 6/18 2/18
Embryonal Carcinoma
Testis 7/7 0 0 6/7 0 4/7 4/7
Mediastinum 3/3 0 0 0 1/3 0 1/3
Testis 19/19 2/19 5/19 6/19 5/19 3/19 14/19
Choriocarcinoma Mediastinum 6/6 6/6 0 0 6/6 0 0
Testis 3/3 3/3 0 0 3/3 0 0
Abbreviations: PLAIt,placental-like alkaline phosphatase; AFP, alpha-fetoprotein; hCG, human [3-chorionicgonadotropin; VIM,vimentin.
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!:
FIGURE 1. Mediastinal seminoma showing paranuclear dot-like CAM 5.2 low-molecular-weight cytokeratin positivity.
FIGURE 3. Strong membrane positivity is seen in this seminoma cell for the CD30 antigen.
The immunohistochemical profile of germ cell tumors in gonadal locations has been extensively studied in the past, often with conflicting results being reported in the literature. 8,~°q5 The immunostaining pattern of extragonadal germ cell tumors, howevm, has not been as extensively investigated. In particular, no comprehensive studies in large series of cases have been available to compare the immunohistochemical profile of the gonadal tumors with those arising in extragonadal locations. We examined 67 mediastinal and 53 testicular germ cell tumors using a panel o f immunohistochemical stains in sections taken from archival, paraffin-
e m b e d d e d material. The most striking differences were f o u n d in the group of seminomas: mediastinal seminomas were characterized by a striking dot-like paranuclear positivity with CAM 5.9 antibodies in 32 of 40 cases, compared with testicular seminomas in which a similar staining pattern was observed in only 5 of 24 cases. Mediastinal seminomas also showed focal cytoplasmic positivity with broad-spectrum keratin antibodies in 97 of 40 cases, whereas testicular seminomas were uniformly negative for this antibody. Stains for PLAP and vimentin were also f o u n d to be more frequently positive in mediastinal than in testicular seminomas. Another interesting finding was the demonstration of focal positivity with CD30 antibodies in isolated t u m o r cells in four cases of seminoma, and in clusters of tumors cells in six cases of yolk sac tumor. No significant differences between the staining patterns of testicular and mediastinal tumors could be identified for any of the other tumor categories in the study, including yolk sac tumor, embryonal carcinoma, and choriocarcinoma. The differences of expression for cytokeratin intermediate filaments between mediastinal and testicular seminomas is of interest and may have some bearing on recent observations regarding the pathogenesis of these lesions. Results of molecular, cytogenetic, and DNA
FIGURE 2. positivity.
FIGURE 4. Yolk sac tumor of the mediastinum showing strong CAM 5,2 low-molecular-weight cytokeratin positivity.
with CAM 5.2 than with broad-spectrum keratin. T h e strongest staining reaction was observed in the cytotrophoblastic components, although syncytiotrophoblastic cells were also positive. All cases in the study also showed strong positivity of the syncytiotrophoblastic c o m p o n e n t for hCG. Stains for AFP, PLAP, vimentin, and CD30 were negative in the tumor cells of all cases tested.
DISCUSSION
Mediastinal seminoma showing focal vimentin
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4 '-t
4
FIGURE 5. Yolksac tumor showing membrane positivity of the tumor cells for CD30.
FIGURE 7. Embryonal carcinoma showing strong membrane and focal dot-like paranuclear positMty for CD30.
ploidy studies appear to indicate that significant differences may exist between mediastinal and testicular seminomas. In a flow cytometric study of mediastinal germ cell tumors, Oosterhuis et a116 were able to demonstrate that most cases o f s e m i n o m a in the mediastinum were diploid, in comparison with testicular seminomas of the adult, which are predominantly aneuploid. In another recent study comparing k-ras-2 gene sequence analysis and expression of p53 between mediastinal and testicular seminomas, we were able to demonstrate the presence of a single-codon 13 k-ras point mutation in 1 of 13 mediastinal seminomas, while the rest of the tumors showed a normal germline configuration. 17 This stands in contrast with most of the previously published studies, which have reported an incidence as high as 40% of K-ras codon-12 point mutations in testicular seminomas. 18,19Additionally, we were able to show that p53 was only weakly and focally expressed in 4 of 13 cases of mediastinal seminoma (31%), as compared with 10 of 13 cases of testicular seminoma (77%). The expression of keratin and vimentin intermediate filaments similarly appears to point to differences between testicular and mediastinal seminomas. Some of
FIGURE6. Embryonal carcinoma showing strong CAM 5.2 low-molecuLar-weight cytokeratin positivity. 740
the early immunohistochemical studies reported in the literature appeared to indicate that seminoma ceils did not contain cytokeratin intermediate filaments. ~°,a2,2°In a study of testicular seminomas using affinity-purified antibodies by indirect immunofluorescence by Denk et al, 13 the authors f o u n d that most of the tumor cells appeared to lack cytokeratin-intermediate filaments, yet most of the cells contained desmosomes, as demonstrated by strong labeling with desmoplakin antibodies, indicative of early or incipient epithelial differentiation. It was further n o t e d that many of the seminoma ceils contained sparse vimentin filaments and that the arr a n g e m e n t and density o f these cytoskeletal intermediate filaments varied with the degree of maturation of the tissue. T h e most regular distribution and intensive staining of cytokeratin and desmoplakin was observed in the more " m a t u r e " tissues. A subsequent study by Niehans et al s in a large series of cases of germ cell tumors using a "cocktail" o f monoclonal cytokeratin antibodies (including CAM 5.2, MAK-6, and AE1/AE3) using the avidin-biotin-peroxidase complex technique f o u n d focal to diffuse keratin positivity in up to 10% of seminomas. Another study by Fogel et a115again demonstrated the heterogeneity of intermediate filament expression in testicular seminoma, with some of the cases showing neither cytokeratin n o r vimentin, some of the cases showing only cytokeratin or vimentin, and more than half of the cases showing co-expression of keratin and vimentin intermediate filaments. Whereas in most of the cases in that study, the cytokeratin subtypes present were mostly of the "simple epithelia" type (CK 8 and 18), in two cases the seminoma ceils also contained cytokeratins 4 and 12, which are normally f o u n d in stratified or complex glandular epithelium, thus denoting a potential for a more advanced and specialized degree of epithelial differentiation. The finding of cytokeratin expression in up to 20% of cases of testicular seminoma in our study is thus in agreement with previous studies reported in the literature for these tumors. The finding of up to 80% o f keratin positivity in mediastinal seminomas in our study, however, requires further explanation. Traditionally, seminomas have been thought to correspond to terminally differentiated germ cell tu-
MEDIASTINAL AND TESTICULARGERM CELL TUMORS (Suster et al)
mors. 21 This view, however, has been challenged in recent years based on the observation of the frequent admixtures and transitions of seminoma with other types of germ cell tumors. 15,2z The demonstration of keratin, vimentin, and desmoplakin immunoreactivity in these tumors, which are markers that are not normally expressed in germ cells, ~3 lends additional support to the notion that seminomas may not represent terminally differentiated neoplasms but are capable of either somatic, embryonal, or trophoblastic differentiation u n d e r certain circumstances. Niehans et al 8 speculated that cytokeratin production by seminoma cells may be one of the earliest indications that such cells are committed toward embryonal, yolk sac, or trophoblastic differentiation, and that expression of these proteins may precede morphological changes detectable at the light microscopic level. The marked differences in expression of these markers between mediastinal and testicular seminoma observed in the current study could thus be attributed to the mediastinal tumors showing a higher degree of differentiation than the testicular neoplasms. This observation is in agreement with the results of DNA ploidy and cytogenetic studies, which all point toward a more stable, mature cellular population in mediastinal seminomas compared with the testicular tumors. 16,17The presence of a more stable, mature, and better differentiated tumor cell population in mediastinal seminoma, as demonstrated by the lower incidence of point mutations, their diploid cell population, low expression of p53, and higher content of cytokeratin and vimentin intermediate filaments, would also explain the better prognosis and lower incidence of metastasis and aggressive behavior of mediastinal semin o m a compared with their testicular counterparts. 24 T h e presence of CD30 positivity in occasional seminoma cells and yolk sac tumors in our study is also of interest. M t h o u g h originally described as an activation marker expressed in Hodgkin's disease and anaplastic large cell lymphoma, recent studies have also demonstrated expression of the CD30 antigen in embryonal carcinomas. ~527 In fact, it has been stated that CD30+ represents a distinctive feature of embryonal carcinoma that may be helpful for the differential diagnosis from other germ cell tumors. 25,27A more recent study, however, was able to detect immunohistochemical staining for CD30 in isolated seminoma cells in 4 of 21 cases of classic testicular seminoma. 2a Our findings confirm these observations and support the close histogenetic relationship that exists between seminoma and embryohal carcinoma. The finding of occasional isolated CD30+ seminoma cells can hardly be regarded as a serious pitfall for the diagnosis of these tumors and is merely of interest for its histogenetic implications. The finding of strong m e m b r a n e and cytoplasmic positivity for CD30 in yolk sac tumor, however, can potentially create serious difficulties for the diagnosis of these tumors. Awareness of this fact is therefore of importance in clinical practice to avoid misinterpreting CD30 positivity in an otherwise typical yolk sac t u m o r for evidence of embryonal carcinomatous differentiation. Such cases should be evaluated within the context of the
morphology and with the aid of an additional panel of immunostains for adequate interpretation. Along similar lines, the strong dot-like cytokeratin positivity observed in mediastinal seminomas, although of limited diagnostic value, may introduce difficulties for diagnosis, particularly in small mediastinoscopic biopsy specimens, in which the cytokeratin positivity could be misinterpreted as indicating the presence of a carcinoma, either primary or metastatic to the thymus. Attention to the distinctive dot-like paranuclear pattern of staining, however, should alert the pathologist to the possibility that she or he is dealing with a mediastinal seminoma, and should p r o m p t the search for immunoreactivity with other more specific antibodies. In summary, the current study appears to indicate the existence of significant differences in immunostaining pattern between mediastinal and testicular seminomas, in particular for cytokeratin and vimentin intermediate filaments. These differences may point toward a more mature cell population with a more advanced stage of differentiation for the tumor cells in mediastinal seminomas. Additionally, the identification of CD30 immunoreactivity in seminoma and yolk sac tumor, in addition to embryonal carcinoma, appears to further highlight the close histogenetic relationship that exists among these tumors.
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13. Denk H, Moll R, Weybora W, et al: Intermediate filaments and desmosomal plaque proteins in testicular seminoma and nonseminomatous germ cell tumors as revealed by immunohistochemistry. Virchows Arch [A] 410:295-307, 1987 14. BenfleyAJ, Parkinson MC, Harding BN, et al: A comparative morphological and immunohistochemical study of testicular seminoma and intracranial germinoma. Histopathology 17:443-449, 1990 15. Fogel M, Lifschitz-Mercer B, Moll R, et al: Heterogeneity of intermediate filament expression in human testicular seminomas. Differentiation 45:242-249, 1990 16. OosterhuisJW, Rammeloo RHU, Cornelisse CJ, et al: Ploidy of malignant mediastinal germ cell tumors. HuM PATHOL 21:729-732, 1990 17. Przygodzki RM, Moran CA, Suster S, et al: Primary mediastinal and testicular seminomas: A comparison of K-ras-2 gene sequence and p53 immunoperoxidase analysis of 25 cases. HuM PATHOL27:975979, 1996 18. MoulJW, Theune SM, Chang EH: Detection ofras mutations in archival testicular germ cell tumors by polymerase chain reaction and oligonucleotide hybridization. Genes Chromosom Cancer 5:109118, 1992 19. Mulder MR Keijzer W, Verbek A, et al: Activated ras oncogene in human seminoma: Evidence for tumor heterogeneity. Oncogene 4:1345-1351, 1989 20. NakagawaY, Perentes E, Ross GW, et al: Immunohistochemical differences between intracranial germinoma and their gonadal
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equivalents: An immunoperoxidase study of germ cell tumors with epithelial membrane antigen, cytokeratin, and vimentin. J Pathol 156:67-72, 1988 21. Teilum G: Classification of endodermal sinus tumor (mesothelioma vitellinum) and so-called "embryonal carcinoma" of the ovary. Acta Pathol Microbiol Scand 64:407-429, 1965 22. Skakkebaek NE, Berthelsen JG: Carcinoma in-situ of the testis and invasive growth of different types of germ cell tumors: A revised germ cell theory. IntJ Androl 4:26-34, 1981 23. Osborn M, Weber K: Tumor diagnosis by intermediate filament typing: A novel tool for surgical pathology. Lab Invest 48:372-394, 1983 24. Moran CA, Suster S, Przygodzki RM, et al: Germ cell tumors of the mediastinum. II. Mediastinal seminoma: A clinicopatholo~cal and immunohistochemical study of 120 eases. Cancer 80:691-698, 1997 25. Pallesen G, Hamilton-Dutoit ST: Ki-1 (CD30) antigen is regularly expressed by tumor cells of embryonal carcinoma. Am J Patho1133:446-450, I988 26. Latza U, Foss H-D, Durkop H, et al: CD30 antigen in embryonal carcinoma and embryogenesis and release of the soluble molecule. AnlJ Patho1146:463-471, 1995 27. Ferreiro JA: Ber-H2 expression in tesficular germ cell tumors. HUM PATHOL25:522-524, 1994 28. Hittmair A, Rogatsch H, Hobisch A, et al: CD30 expression in seminoma. HUN PATttOL27:1166-1171, 1996