Placental alkaline phosphatase immunohistochemistry of intratubular malignant germ cells and associated testicular germ cell tumors

Two hundred three testicular germ cell tumors were studied immunohistochemically for the presence of placental alkaline phosphatase (PLAP). Special emphasis was placed on the pattern and incidence of positive staining of intratubular malignant germ cells (ITMGCs) adjacent to tumors. 99% of cases with adjacent ITMGCs showed a positive staining reaction in some or all IT-MGCs present. Other germ cell elements showed at least a focal positive staining reaction in the following proportions: seminomas, 96%; embryonal carcinomas, 96%; yolk sac tumors, 25%; mature teratomas, 5%; immature teratomas, 4%; choriocarcinomas, 45%; and syncytiotrophoblasts, 43%. The staining pattern for seminomas tended to be diffuse, whereas for embryonal carcinomas the staining pattern was more focal. Yolk sac tumors stained inconsistently for PLAP and a positive reaction was limited to a small percentage of cells. Syncytiotrophoblasts, singly or in choriocarcinomas, also showed variable positivity. These results corroborate the fact that PLAP is a sensitive marker for ITMGC, seminoma, and embryonal carcinoma.     

D2-40 immunohistochemistry in the differential diagnosis of seminoma and embryonal carcinoma: a comparative immunohistochemical study with KIT (CD117) and CD30.

The distinction between seminoma and embryonal carcinoma based on morphology alone can sometimes be problematic, requiring the use of immunohistochemistry to facilitate diagnosis. D2-40 is a monoclonal antibody that reacts with an oncofetal antigen expressed by fetal germ cells and testicular germ cell tumors. The diagnostic value of D2-40 immunohistochemistry in distinguishing seminoma from embryonal carcinoma has not been determined. D2-40 immunoreactivity was evaluated in a series of testicular germ cell tumors and compared with that of KIT (CD117) and CD30, to assess the relative utility of this marker in discriminating between seminoma and embryonal carcinoma. Forty testicular germ cell neoplasms were examined, which included 19 seminomas, three embryonal carcinomas, three teratomas, one yolk sac tumor, and 14 mixed germ cell tumors. The 14 cases of mixed germ cell tumors contained components of seminoma (n=7), embryonal carcinoma (n=11), teratoma (n=10), yolk sac tumor (n=2), and choriocarcinoma (n=1). All cases of pure seminoma and the seminomatous components of mixed germ cell tumors exhibited positive immunoreactivity for D2-40. Focal positivity for D2-40 was also observed in 29% of the embryonal carcinoma samples. D2-40 immunoreactivity in seminomas was characterized by diffuse membrane staining, whereas for embryonal carcinomas, staining was focal and distributed along the apical surfaces of the neoplastic cells. Immunohistochemical staining for KIT was observed in 92% of the seminoma samples and in none of the embryonal carcinomas. Conversely, CD30 expression was identified in 93% of the embryonal carcinoma samples and in none of the seminomas. Other germ cell components showed no immunoreactivity for D2-40, KIT, or CD30. KIT and CD30 are effective immunohistochemical markers in separating seminoma from embryonal carcinoma. Although a highly sensitive marker for seminomas, D2-40 positivity was also observed in a subset of embryonal carcinomas, thus limiting the utility of this antibody for discriminating between these two malignancies.     

Evaluation of cyclin expression in testicular germ cell tumors: cyclin E correlates with tumor type, advanced clinical stage, and pulmonary metastasis.

The measurement of proliferative index has yielded promising yet conflicting results in the evaluation of testicular tumors. We have examined the role of Ki-67, along with the cyclins A and E in testicular tumorigenesis. We compared the immunoreactivity of 20 pure seminomas with 20 mixed germ cell tumors composed predominantly of embryonal carcinoma with a variety of proliferation markers, including Ki-67, cyclin A, and cyclin E. All 40 tumors stained for Ki-67, and 19 of 20 (95%) seminomas and 18 of 20 (90%) embryonal carcinomas stained positively for cyclin A. Cyclin E stained 14 of 19 (74%) of the embryonal carcinomas and only 4 of 20 (20%) of the seminomas (Fisher's exact two-tailed test, P = .0012). There was a trend toward larger tumor size for cyclin E-positive seminomas (median, 5.92 cm versus 3.96 cm; P = .08), although the same correlation was not significant in embryonal carcinomas. For both seminomas and embryonal carcinomas, staining with cyclin E did not correlate with the presence of lymphovascular invasion or capsular invasion. However, patients who had cyclin E-positive tumors presented with higher clinical stage (P = .0015). In addition, pulmonary spread in embryonal carcinomas (four patients) and seminomas (one patient) occurred only in patients whose tumors were cyclin E positive (P = .014). Although Ki-67 and cyclin A offer little prognostic information in testicular germ cell tumors, cyclin E immunoreactivity correlates with tumor type and is strongly predictive of distant tumor spread.     

Frequent Fas gene mutations in testicular germ cell tumors

The Fas (Apo-1/CD95)/Fas ligand (L) system is involved in cell death signaling, and has been suggested to be important for the regulation of germ cell apoptosis in the testis. Mutations of the Fas gene may result in accumulation of germ cells and thus might contribute to testicular carcinogenesis. The open reading frame of Fas cDNA was examined in 24 cases of testicular germ cell tumors (TGCTs), comprised of 19 pure histological type (15 seminomas, 3 embryonal carcinomas, 1 immature teratoma) and 5 mixed-type tumors. Mutations of the Fas gene were found in nine (37.5%) of these cases. Each lesion with a homogeneous histological picture was selectively microdissected using a laser capture microdissection method: samples consisted of 18 lesions from seminomas, 7 embryonal carcinomas, 4 immature teratomas, 2 choriocarcinomas, and 1 from a yolk sac tumor. Microdissected genomic DNA was examined to determine which mutations were derived from which kind of histological lesion. Eleven mutations were detected in 10 TGCT lesions from nine cases, but none were found in benign lesions. All were point mutations, and eight missense mutations occurred in exon 9 encoding the core protein of the death domain essential for apoptotic signal transduction. Three were silent mutations. Mutations were found in the seminoma (27.8%) and embryonal carcinoma lesions (62.5%), but none were found in the one yolk sac tumor, two choriocarcinomas, or four immature teratoma lesions. Each seminoma and embryonal carcinoma lesion found in the same case had a different type of Fas mutation from the others. Mouse T-cell lymphoma cells transfected with missense mutated genes were resistant to apoptosis induced by anti-Fas antibody, indicating these to be loss-of-function mutations. These findings suggested a role of Fas gene mutations in the pathogenesis of TGCTs.     

Intratubular malignant germ cells in testicular biopsies: clinical course and identification by staining for placental alkaline phosphatase

This study was undertaken to determine the clinical course of patients with intratubular malignant germ cells (ITMGCs) and to evaluate the reliability of placental alkaline phosphatase (PLAP) for detection of these cells. Eight patients with ITMGCs in testicular biopsy were followed. Four patients received no further immediate treatment. Two of these showed no evidence of disease after 190 and 132 mo; one developed seminoma at 38 mo, and one developed seminoma of the contralateral testis in 61 mo. The remaining four patients underwent immediate orchiectomy. Orchiectomy findings were invasive seminoma in three testes, intratubular seminoma in one, and residual ITMGCs in one testis (one patient had bilateral orchiectomy). Also studied were two testicular biopsies from patients with known retroperitoneal germ cell tumors, respectively, seminoma and teratoma. Both had ITMGCs in their testes. Immunoperoxidase stains for PLAP gave a positive result in all biopsies showing intratubular malignant germ cells. PLAP was not demonstrated in spermatogonia in 471 control biopsies which did not show germ cell neoplasia. Two other patients with incidental seminoma on biopsy for infertility are discussed. These results show that ITMGCs show a high rate of progression to invasive disease, but can show an indolent course. PLAP is a sensitive and specific marker for ITMGC, facilitating diagnosis.     

MAGE-1 and MAGE-3 tumor rejection antigens in human germ cell tumors.

The MAGE-1 and MAGE-3 genes are members of the melanoma antigen-encoding gene family. These genes encode for HLA-restricted tumor-associated rejection antigens recognized by cytotoxic T lymphocytes. MAGE-1 and MAGE-3 gene expression has been identified in a number of human malignancies, and MAGE-1 and MAGE-3 antigenic peptides are potential targets for tumor-specific immunotherapy. The only normal tissues known to express these genes are testicular germ cells and placental tissue. The objective of this study was to examine MAGE-1 and MAGE-3 antigens immunohistochemically in testicular germ cell tumors, including seminoma, a germ cell tumor frequently associated with a lymphoid infiltrate. Forty-three germ cell tumors (24 seminomas, six embryonal carcinomas and 13 mixed germ cell tumors), and 10 Leydig cell tumors were selected for study, and standard immunohistochemical techniques were used on formalin-fixed paraffin-embedded tissues using mouse monoclonal antibodies to MAGE-1 (clone M454) and MAGE-3 (clone 57B) antigens. MAGE-1 antigen was identified in 16.6% of seminomas. No embryonal carcinomas, yolk sac tumors, or teratomas contained MAGE-1 protein. MAGE-3 antigen was identified in 41.8% of seminomas, and this protein was not identified in embryonal carcinomas, yolk sac tumors, or teratoma. Spermatogonia and primary spermatocytes contained MAGE-1 and MAGE-3 antigen, and more mature forms, including spermatids, were weakly positive to negative. Leydig cell tumors were negative for MAGE-1 and MAGE-3. In seminoma, the presence of MAGE-1 and MAGE-3 antigens did not correlate with tumor size, tumor stage, the presence of a lymphoid infiltrate, or patient outcome. The low frequency of MAGE-specific HLA alleles in the population, the loss of the HLA class I antigens in neoplastic germ cells, and the finding that the majority of seminomas and all non-seminomatous germ cell tumors lacked MAGE-1 and MAGE-3 antigenic peptides indicate that immunotherapy directed towards MAGE-1 and MAGE-3 antigen is not a likely treatment option for seminoma and nonseminomatous germ cell tumors. The significance of MAGE-1 and MAGE-3 proteins in normal spermatogonia and primary spermatocytes will require additional study.     

Alpha-fetoprotein in germ cell tumors. An immunohistochemical study

Total 40 cases of testicular and ovarian germ cell tumors and one case of extragonadal germ cell tumor were studied for the presence of alphafetoprotein (AFP) by indirect immunoperoxidase technique. All seminomas (7 cases) and teratomas (13 cases) were negative for AFP; while 85% of the pure embryonal carcinomas, (E.C.) all pure yolk sac tumors (Y.S.T.) (7 cases) and all embryonal carcinoma and yolk sac components in mixed tumors were AFP positive. Immunostaining of tumor marker appeared to help only in differentiating seminomatous and nonseminomatous tumors and hence does not provide any additional information for classification of these tumors.     

Immunohistochemical localization of lactate dehydrogenase isoenzyme 1 in germ cell tumors of the testis

In this study, antiserum to lactate dehydrogenase isoenzyme 1 (LD 1) was used to determine immunohistochemical patterns of localization in a variety of germ cell neoplasms of the testis. All 29 seminomas and teratocarcinomas and four of seven embryonal carcinomas were positive for LD 1. These results suggest that elevated serum LD 1 levels noted in patients with germ cell neoplasms may be derived directly from neoplastic tissue and explain the relationship between serum LD 1 levels and tumor burden. A variety of neoplasms from other organs also were evaluated in order to determine the specificity of LD 1 staining for testicular tumors.     

Monoclonal antibody 43-9F: an immunohistochemical marker of embryonal carcinoma of the testis.

Discrimination between different types of germ cell tumours may be difficult in routine histological preparations. Additionally, none of the established immunohistochemical markers is completely reliable in diagnosis of embryonal carcinoma. A pilot study indicated that monoclonal antibody 43-9F--a marker of carcinoma-in-situ germ cells--may also react with embryonal carcinoma of the testis. In order to elucidate the applicability of 43-9F in diagnosis of embryonal carcinoma, 42 consecutive testicular germ cell tumours were tested immunohistochemically. Among the 42 tumours, 23 were seminomas and 19 were non-seminomas with seminomatous components in seven of them. Embryonal carcinomas were found in 15 tumours, two being of pure type and the remaining 13 a part of mixed tumours. Additionally, the material included 11 teratomas, nine yolk sac tumours and one choriocarcinoma. Immunohistochemical stainings were performed with 43-9F and additionally with antibodies against placental-like alkaline phosphatase, cytokeratins, alpha-foetoprotein and human chorionic gonadotropin. Using 43-9F a strong colour reaction was found in 13 of the embryonal carcinomas, whereas the reaction was moderate in the remaining two cases. A weak positive reaction was found in six seminomas and the remaining 24 did not react at all. 43-9F exhibited a positive reaction in four of 11 teratomas. The reactivity was generally weak with some focal areas with strong staining. In five cases the yolk sac tumour elements did not stain with this monoclonal antibody. The reaction was weak in three cases and in only one case was the staining intensity scored as moderate. Finally, no reaction was found in the choriocarcinoma element. Compared to the other antibodies tested, including the antibody against cytokeratins, in embryonal carcinoma immunohistochemical staining with 43-9F was more specific, stronger and more constantly expressed. The monoclonal antibody 43-9F may be of value in histological diagnosis of germ cell tumours. Additionally, the study confirmed the pathogenetical link between pre-invasive carcinoma in situ and embryonal carcinoma.     

Nucleolar organizer regions in seminoma and intratubular malignant germ cells

Nucleolar organizer regions (NORs), as demonstrated by a silver-colloid staining technique, have been counted in 71 primary testicular seminomas (typical seminoma (TS) 31, high mitotic index seminoma (HMIS) 24, and spermatocytic seminoma (SS) 16) and ten seminomas metastatic to retroperitoneal lymph nodes. Mean NOR counts were 14.36 for TS; 17.66 for HMIS; 10.89 for SS; and 17.70 for metastatic seminoma. Analysis of data using Student's unpaired t-test showed a significant difference between the NOR counts obtained from TS, HMIS, and SS. Furthermore, there was a statistically significant difference between NOR counts in metastatic seminoma when compared with TS and SS but not HMIS. The association between tumor proliferation rates and intranuclear NOR numbers is discussed. In addition to a numerical variation, the NOR distribution throughout the nucleus was noted to be different in SS when compared with the other varieties of seminoma studied. The pattern observed had some features similar to those seen in cells of the spermatogenic series. The NOR technique was also applied to 19 cases of intratubular malignant germ cells (ITMGC). It was shown that these malignant cells were easily identified using this staining method and that the NOR distribution was similar to that seen in TS and HMIS. The mean NOR count in ITMGC was 16.41. This was significantly different from that of TS but not HMIS.     

Ultrastructural characteristics of seminoma and embryonal cancer of the testis

Twenty-three germ cell tumors of the testis were investigated. Electron microscopic examinations of four seminomas and four embryonal carcinomas revealed cells of different degrees of differentiation. Tumor cells of seminoma acquired the ultrastructural features of the cells of developing seminal epithelium whereas in embryonal carcinoma there was a pronounced tendency of formation of mature tissues of teratoma. These data confirmed the cytogenetic resemblance of seminoma and embryonal carcinoma originating from the seminal epithelium which could undergo malignization at different stages of development. The latter predetermined diverse direction of differentiation in germ cell tumors of the testis.     

Loss of Fhit expression in testicular germ cell tumors and intratubular germ cell neoplasia.

The FHIT gene, located at human chromosome 3p14.2, is frequently deleted in a number of human cancers, and interstitial deletions at this site were recently described in a significant proportion (41%) of testicular germ cell tumors. We studied the expression of Fhit protein in the progression and differentiation of testicular germ cell tumors to further elucidate its role in this type of malignancy. Forty-five patients with testicular germ cell tumors and intratubular germ cell neoplasia (identified in 42/45 cases) were included in the study. Immunohistochemical staining with polyclonal rabbit IgG antibody to Fhit (ZR44, Zymed Laboratories) on formalin-fixed, paraffin-embedded tissues was used. Fhit was constitutively expressed in germ cells, Sertoli cells, and Leydig cells. All 42 cases of intratubular germ cell neoplasia revealed no expression of this protein. No expression of Fhit was observed in any case of pure seminoma or in the seminomatous component of mixed germ cell tumors. Unexpectedly, Fhit expression was frequently (16/18) observed in the glandular tissue of mature teratomatous component of mixed germ cell tumors, despite the absence of Fhit in the intratubular germ cell neoplasia, the presumed precursor lesion. The loss of Fhit expression is a consistent characteristic of intratubular germ cell neoplasia, which suggests a potential role in a maturation/differentiation defect early in the development of testicular germ cell tumors. Likewise, the lack of expression in seminomas is supportive of this view. However, re-expression of Fhit in well-differentiated glandular epithelium of teratomatous component of mixed germ cell tumors suggests that there is no loss of FHIT gene in this subset of neoplasia but rather that Fhit protein expression is differently regulated through the phases of germ cell tumor progression.     

A monoclonal antibody as a marker for carcinoma in situ germ cells of the human adult testis

Carcinoma in situ of the testis (CIS) is a precursor of invasive testicular germ cell tumours. The diagnosis of CIS is however often missed when conventional histological techniques are used. No specific immunological marker for CIS germ cells of the testis has been demonstrated previously. A novel monoclonal antibody, M2A, reacting with malignant germ cells of seminomas has recently been developed. Using the immunoperoxidase reaction on tissue sections, we tested the reactivity of M2A with CIS germ cells of the human adult testis. Positive reaction was found in 19 of 20 testicular specimens showing CIS, whereas no staining was found in 39 testicular biopsies without CIS. Thus, M2A may serve as a diagnostic marker in detection of CIS germ cells.     

Epidermal growth factor receptor is a marker for syncytiotrophoblastic cells in testicular germ cell tumors

The epidermal growth factor receptor (EGFR) has been implicated in the pathogenesis, therapy and prognosis of various tumor types. The aim of this study was to investigate EGFR expression in a large series of testicular germ cell tumors (TGCTs). A total of 88 TGCTs (37 of pure type and 51 of mixed type) comprising a total of 44 seminoma, 49 embryonal carcinoma, 32 yolk sac tumor, 28 teratoma and 7 choriocarcinoma components were immunostained for EGFR. EGFR reactivity was observed in the stromal cells of embryonal carcinoma (29%) and in epithelial compartments of teratoma (71%). In addition, EGFR staining was consistently detected in syncytiotrophoblastic cells of choriocarcinoma, seminoma, embryonal carcinoma and yolk sac tumor components. EGFR staining, similar to beta-human chorionic gonadotropin (HCG) immunohistochemistry, was efficiently able to identify syncytiotrophoblastic cells in TGCTs. This study shows that EGFR is expressed in a subset of testicular germ cell tumors and suggests that EGFR may be a useful marker for syncytiotrophoblastic cells.