Immunohistochemical localisation of alpha-fetoprotein (AFP) in germ cell tumours: evidence for AFP production by tissues different from endodermal sinus tumour

20 germ cell tumours have been studied with respect to the presence of alpha-fetoprotein (AFP), using the peroxidase-antiperoxidase (PAP) technique. 6 out of 20 tumours contained elements of endodermal sinus tumour (EST) and were AFP positive. 16 tumours were diagnosed either as pure embryonal carcinomas (6) or as mixed germ cell tumours, containing elements of embryonal carcinoma (10). In 3 of these 16 tumours AFP was localised definitely in the embryonal carcinoma cells; in an additional 6, AFP was also detected but it could not be decided whether AFP was present in embryonal carcinoma cells or in EST cells during early differentiation. In 2 of 7 immature teratomas, AFP was shown to be present in cylindric epithelia. All seminomas (4) studied were AFP-negative. These results show that AFP, which occurs regularly in EST, may also be present in embryonal carcinomas as well as in immature teratomas. Thus, it seems that the immunohistochemical demonstration of AFP by the PAP technique is a suitable method of identifying late stages of embryonal carcinoma or early stages of endodermal sinus tumour during the process of differentiation.     

Protein overexpression and gene amplification of epidermal growth factor receptor in adult testicular germ cell tumors: Potential role in tumor progression

Little is known about the pathologic significance of epidermal growth factor receptor (EGFR) expression in malignant testicular germ cell tumors (TGCTs) in adults. From the primary tumor sites of a cohort of 110 TGCT cases, we obtained 209 histologically distinct components: 53 intratubular germ cell neoplasia unclassified (IGCNU) lesions, 83 seminomas (66 pure‐form seminomas and 17 seminoma components in the mixed‐form with nonseminomatous TGCTs), 27 embryonal carcinomas, eight choriocarcinomas, 18 yolk sac tumors, and 20 immature teratomas. Samples were analyzed for expression of EGFR protein and EGFR gene amplification by immunohistochemistry and fluorescence in situ hybridization (FISH), respectively. Overexpression of the EGFR protein was detected in 28% of seminomas (27% in the pure‐form and 29% in the mixed‐form), 11% of embryonal carcinomas, 88% of choriocarcinomas, 44% of yolk sac tumors, and none of the IGCNU lesions or immature teratomas. A higher copy number (≥4 copies per cell) and amplification of the EGFR gene were detected in 20% and 10% of seminomas, 13% and 0% of embryonal carcinomas, 71% and 60% of choriocarcinomas, 15% and 8% of yolk sac tumors, and none of the IGCNU lesions or immature teratomas, respectively. Both higher copy number and amplification of the EGFR gene were positively correlated with immunohistochemical overexpression of EGFR protein (each P < 0.0001). These results suggest that overexpression of EGFR protein and increased copy number or amplification of the EGFR gene occur relatively frequently in primary TGCTs, and may play roles in the formation of invasive cancer and in the progression, especially morphological evolution, of tumors. (Cancer Sci 2010)     

Differentiation patterns of testicular germ-cell tumours as revealed by a panel of monoclonal antibodies

A panel of monoclonal antibodies against different keratin polypeptides, epithelial glycoproteins, placental alkaline phosphatase and collagen type IV was used to evaluate immunohistochemically the expression of the target antigens in 30 different human testicular germ-cell tumours of various types. Antikeratin antibodies detecting markers of different routes of epithelial differentiation revealed remarkable similarity of differential expression of various keratins in epithelial structures of teratomas and combined tumours as compared with normal human epithelial tissues. A considerable proportion of embryonal carcinoma cells stained positively for keratins 8, 18 and 19, while a minor subpopulation of tumour cells in embryonal carcinomas, some seminomas and many atypical intratubular cells expressed keratins 8 and 18 but usually lacked keratin 19. Antibody RICEO-MFG-06.3, specific for epithelial glycoproteins, gave negative results with seminomas as opposed to positivity in all but two nonseminomatous tumours. All but two neoplasms showed positivity for placental alkaline phosphatase, thus supporting its reliability as a marker of germ-cell tumours. It is concluded that the monoclonal antibody RICEO-MFG-06.3 and especially the keratin-19-specific antibodies BA16 and BA17 can be helpful in distinguishing embryonal carcinoma from seminoma and, together with antibodies to other keratins, in the study of the origin and histogenesis of testicular germ-cell tumours.     

Malignant ovarian tumours in childhood in Britain, 1962-78

The files of the Childhood Cancer Research Group and of the Oxford Survey of Childhood Cancers were scrutinized for all the ovarian neoplasms registered in England, Scotland and Wales in children under age 15 years throughout the period 1962-78. Among 172 cases confirmed as malignant ovarian tumours, 145 (84%) were tumours of germ cell origin (54 dysgerminomas, 36 malignant teratomas, 26 endodermal sinus tumours, 4 embryonal carcinomas, 2 pure choriocarcinomas, 20 mixed germ cell neoplasms, 3 gonadoblastomas), 13 (8%) were epithelial carcinomas (3 serous or undifferentiated, 10 mucinous), 9 (5%) were sex-cord stromal tumours (3 granulosa cell, 3 Sertoli-Leydig, 3 unclassified) and 5 (3%) were other miscellaneous tumour types. Less than 10% of the neoplasms occurred at age less than 5 years, approximately 20% from 5-9, and greater than 70% from 10-14 years. Germ cell neoplasms of greater malignancy (immature teratomas, endodermal sinus tumours) occurred in a significantly higher proportion at younger age (less than 10 years) than dysgerminomas (P = 0.01). The overall incidence (approximately 1.7 cases per 10(6) per annum) did not show any noticeable trend over the 17-year period considered. The clustering of two confined cases and, possibly, a third case, of germ cell neoplasms in three generations of the same family pointed to a genetic component in the aetiology of some of these neoplasms. A large number of sex related and mental or neurological abnormalities was also reported in case children. The 10-year survival rates, determined by the life-table method were: epithelial carcinomas 73%, sex-cord stromal tumours 44%, dysgerminomas 73%, malignant teratomas 33%, endodermal sinus tumours 39%, embryonal carcinomas 25%, other germ cell neoplasms 30% and gonadoblastomas 100%. Apart from cell-type, factors associated with prognosis were clinical stage (in all types), size and degree of histological differentiation (in malignant teratomas, but only when stage was not allowed for). The adoption of efficacious polychemotherapy regimens completely changed the prognosis of germ cell tumours other than dysgerminomas (from 29% to greater than 85% disease-free survivors in the present series).     

DNA topoisomerase I and II expression in drug resistant germ cell tumours

A small number of testicular germ cell tumours are refractory to current chemotherapy regimens. DNA topoisomerase I is the target for several new drugs and a potential candidate treatment for chemorefractory germ cell tumours. DNA topoisomerase II alpha is the target for etoposide, which is currently used regularly in germ cell tumour treatment. The expression of DNA topoisomerase I and II alpha were therefore assessed immunohistochemically in a range of testicular tumours, especially those with persistent malignant elements on retroperitoneal lymph node dissection. Pre-chemotherapy orchidectomy specimens were matched with post-chemotherapy retroperitoneal lymph node dissections to examine changes in expression. There was considerable variation in the expression of topoisomerase I in different tumour types. Both yolk sac tumours and teratoma, mature showed universal expression of topoisomerase I, while 38% of seminomas and 30% of embryonal carcinomas were positive. Strong topoisomerase II alpha expression was found in embryonal carcinoma. There was a negative correlation between topoisomerase I and II alpha expression (P=0.004) and downregulation of topoisomerase II alpha after chemotherapy (P=0.02). Topoisomerase I expression appears to increase in those cases with residual teratoma, mature, but is largely unchanged in those cases remaining as embryonal carcinoma. These results suggest that topoisomerase I inhibitors may be useful in chemorefractory germ cell tumours, especially yolk sac tumours and where there are unresectable residual teratoma, mature deposits.     

Involvement of dysadherin and E-cadherin in the development of testicular tumours

Testicular neoplasms are comprised of a variety of histologically different forms, and their pathogenesis has not been elucidated. Dysadherin is a recently described cell membrane glycoprotein, which has an anticell-cell adhesion function and downregulates E-cadherin. In this study, we examined immunohistochemically the expression of E-cadherin and dysadherin in 120 testicular neoplasms (37 seminomas-26 classic, five spermatocytic and six anaplastic-, 45 embryonal carcinomas, 10 mixed germ cell tumours, two yolk sac tumours, 10 mature and eight immature teratomas and eight non-Hodgkin B-cell lymphomas), clinical stage I. The intensity, the expression pattern and the percentage of neoplastic cell staining was recorded and correlated with the histologic type and vascular/lymphatic invasion. Dysadherin was not expressed in non-neoplastic germ cells, neither in CIS/ITGCNU, but it was highly expressed in all types of germ cell tumours, that demonstrated either embryonic phenotype or somatic differentiation, in most terminally differentiated neoplasms, and in all lymphomas. Dysadherin expression did not correlate with vascular invasion. Increased dysadherin expression was correlated with aberrant E-cadherin expression in most tumours. In 17% of embryonal carcinomas colocalisation of dysadherin and membranous E-cadherin staining was noted. This is the first report on dysadherin expression and its association with E-cadherin in testicular tumours. Since dysadherin is not normally expressed in non-neoplastic testis, it is conceivable that it plays a role in the neoplastic transformation of germ cells. In testicular tumours, as in other neoplasms, dysadherin downregulates E-cadherin expression, at least in part.     

p53 protein alterations in human testicular cancer including pre-invasive intratubular germ-cell neoplasia.

Expression of the p53 oncoprotein was examined in a wide range of primary human testicular germ-cell tumours using a new mouse monoclonal antibody (MAb) BP53-11 raised and characterized in this study, in parallel with a polyclonal rabbit antiserum CM-1. Immunohistochemistry on paraffin sections showed positive nuclear reaction in at least a fraction of malignant cells in 90 (84%) out of 107 cases studied. Aberrant accumulation of the p53 protein was found among testicular tumours of all major histological types, although generally a higher percentage of positive cases and a higher proportion of p53 over-expressing nuclei within individual lesions was observed in embryonal carcinomas when compared with seminomas. The typical heterogeneous staining pattern characteristic of histological specimens was also found in a cultured cell line derived from a human embryonal carcinoma. In contrast to immunohistochemically undetectable levels in normal testes and morphologically normal tissue areas in the tumour-bearing testes, the accumulation of the p53 protein was clearly identified in a high proportion (59% of cases) of the pre-invasive lesions with positive atypical intratubular germ cells often found in the tissue adjacent to invasive tumours. Altered expression of the p53 protein is therefore a unifying feature of the majority of invasive male germ-cell tumours and the change resulting in high levels of p53 appears to be a relatively early step in the human testicular cancer pathogenesis.     

Biologie und Pathologie von Keimzelltumoren des Hodens

Germ cell tumours constitute about 90% of testicular tumours and their incidence has increased in recent decades in Europe. The tumorigenesis of germ cell tumours shows remarkable similarities to embryogenesis and is discussed from a stem cell point of view. Invasive germ cell tumours develop from intratubular germ cell neoplasia and differentiate into seminomas and non-seminomas. The embryonal features of the persistent and neoplastic germ cells are transmitted to the invasive germ cells. Germ cell tumours display a broad histological variety resulting in a rather complicated WHO classification with multiple subtypes.     

Cell cycle regulators in testicular cancer: loss of p18INK4C marks progression from carcinoma in situ to invasive germ cell tumours

Cell cycle regulators govern cellular proliferation, modulate differentiation and, when defective, contribute to oncogenesis. Here, we examined expression of cyclins A, B1 and E, and cyclin-dependent kinase (CDK) inhibitors p18INK4C (p18), p21WAF1/Cip1 (p21) and p27KiP1 (p27), in normal human adult testis (n = 5), and 53 testicular tumours, including 23 carcinomas in situ (CIS) and 30 germ cell tumours (GCTs). Immunohistochemical analysis revealed a correlation between proliferation and abundance of the cyclin proteins, and abundant p18 and the lack of p21 and p27 in normal spermatogenesis. Expression of p21 and/or p27 was induced in some differentiated structures seen in teratomas, and was recapitulated in cell culture, using human NTera2/D1 teratocarcinoma cells induced to differentiate into neurons. CIS lesions showed abundant p18, low cyclin E, and moderate p27, in contrast with most invasive seminomas and embryonal carcinomas with very low-to-negative p18, often elevated cyclin E, and, unexpectedly, sustained or increased p27. Our results suggest increased abundance of cyclin E, and particularly downmodulation or loss of p18INK4C as the features that correlate with progression from CIS to invasive germ cell tumours of the human testis.     

Teratocarcinoma stem cells

Human teratocarcinomas or mixed germ cell tumours are histologically composed of diverse tissues corresponding to somatic and extraembryonic (trophoblastic and yolk sac) like cells, as well as malignant stem cells. In typical teratocarcinomas these stem cells correspond to embryonal carcinoma cells, ie developmentally pluripotent cells equivalent to embryonic cells from the early stages of development. These cells have the capacity to differentiate and give rise to non-proliferating terminally differentiated tissue. Occasionally embryonal carcinoma cells can give rise to more differentiated stem cells which have the phenotype and the restricted developmental potential of choriocarcinoma and yolk sac carcinoma cells, or less commonly to somatic cell malignancies, indistinguishable from typical carcinomas, sarcomas, melanoma or lymphomas. Malignant transformation of benign somatic tissues in teratomas can also give rise to malignant stem cells, which all have a somatic cell phenotype. The biology and the clinical presentation as well as the response to chemotherapy of germ cell tumours depend on the nature of stem cells that form their proliferative compartment and account for the malignancy of these tumours.     

Human germ cell tumours: expression of gamma-glutamyl transpeptidase and sensitivity to cisplatin.

Previous studies have shown that the enzyme-glutamyl transpeptidase (GGT) is essential for the nephrotoxicity of cisplatin. This study was designed to determine whether GGT activity is necessary for the therapeutic effect of the drug. The relationship between GGT expression and clinical response to platinum-based chemotherapy was examined in 41 human germ cell tumours. Sections of formalin-fixed, paraffin-embedded tumours were immunohistochemically stained with an antibody directed against human GGT. There was no expression of GGT in any of the 17 seminomas or four dysgerminomas; whereas, 12/12 ovarian yolk sac tumours and 4/4 embryonal carcinomas of the testis were GGT-positive. In stage I tumours fewer tumour cells expressed GGT than in later stage tumours. In four germ cell tumours of mixed histology, the seminomatous and dysgerminoma areas were GGT-negative while the areas of the tumour with yolk sac or embryonal histology contained GGT-positive tumour cells. The patients with seminomas or dysgerminomas who were treated with cisplatin-based chemotherapy, all had a complete response despite the absence of GGT expression in these tumours. Fifteen of the 16 patients with yolk sac or embryonal carcinomas received cisplatin-based chemotherapy following surgery. Twelve had a complete response, while three failed to respond to platinum-based therapy. There was no correlation between the level of GGT-expression and response to therapy in this group. Three of the four patients with tumours of mixed histology were treated with cisplatin-based therapy, and had a complete response. Therefore, expression of GGT is not necessary for the therapeutic effect of cisplatin in germ cell tumours. The results from this study suggest that systemic inhibition of GGT would inhibit the nephrotoxic side-effect of cisplatin without interfering with its activity towards germ cell tumours.     

Immunohistochemical heterogeneity of type 1 blood group antigen expressions in testicular germ cell tumors

The immunohistochemical expression of type 1 blood group antigens (type 1 BGAs) was analyzed for 30 cases of testicular germ cell tumors (TGCTs), using monoclonal antibodies against DU-PAN-2, CA19-9, Lewis(a) (Le(a)), and Lewis(b) (Le(b)). DU-PAN-2 was expressed very frequently in all of the embryonal carcinomas (ECs). CA19-9 expression was demonstrated in 53% of ECs, but the number of positive cells was generally smaller than that for DU-PAN-2. CA19-9-negative ECs tended to show a higher number of DU-PAN-2-positive cells compared to CA19-9-positive ECs, and ECs in which DU-PAN-2 was more strongly expressed showed a relatively frequent expression of CA19-9. In 36% of seminomas and 56% of yolk sac tumors (YSTs), DU-PAN-2 was weakly expressed, and the positive cells were few in number. Little or no expression of CA19-9 was demonstrated in seminomas and YSTs. Regarding Le(a) and Le(b), the expressions were found to be limited to teratomas at a frequency of 57% and 86%, respectively, with the exception of one EC positive for Lea and one YST positive for Leb. Eighty-six percent of teratomas showed expressions of DU-PAN-2 and CA19-9. DU-PAN-2 was also seen in some intratubular malignant germ cells. The antibodies used were all negative for choriocarcinomas, syncytiotrophoblastic giant cells, and normal testicular tissues. The antigen expressions were predominantly observed on the surface of tumor cells developing luminal structures. In conclusion, although CA19-9 was relatively specific for ECs, it should be emphasized that ECs were rather characteristic of extensive DU-PAN-2 expression. Particularly in CA19-9-negative ECs, a combined analysis of DU-PAN-2 and CA19-9 would be helpful in confirming the histopathologic diagnosis of TGCTs. The clinical significance of DU-PAN-2 in ECs as a tumor marker remains to be clarified. Le(a) and Le(b) expressions were thought to be related to the differentiation or maturation rather than to the malignant transformation in TGCTs.     

Undifferentiated intratubular germ cell tumor of the testis: light and electron microscopic study of a unique case.

A 34-year-old white man was found to have a nodule on the surface of right testis which turned out to be metastatic embryonal carcinoma within the tunica albuginea. Examination of the orchiectomy specimen revealed extensive undifferentiated intratubular germ cell tumor (carcinoma in situ) with foci of intratubular embryonal carcinoma and seminoma. Ultrastructure of the cells comprising the carcinoma in situ was similar to that of undifferentiated germ cells. The development of intratubular embryonal carcinoma and seminoma in a testis with extensive carcinoma in situ lends further support to the concept that carcinoma in situ or the testis represents an undifferentiated germ cell tumor from which other types of germ cell tumors of the testis may evolve.     

Telomerase activity in germ cell cancers and mature teratomas.

BACKGROUND: An inverse relationship has been reported between the presence of telomerase enzymatic activity and the induction of differentiation in human tumor cell lines. Male germ cell tumors represent an attractive clinical model to assess this relationship further because high telomerase activity is present in normal germ cell progenitors and in embryonal carcinomas that can differentiate into mature teratomas. To investigate how telomerase activity and the differentiation state of germ cell tumors are related, telomerase activities and telomere lengths were measured in benign testicular tissues, germ cell cancers, and mature or immature teratomas. METHODS: By use of a modified telomeric repeat amplification protocol (TRAP) assay, telomerase activity was measured in four specimens of benign testicular tissue, in 27 germ cell cancers, in seven mature teratomas, and in one immature teratoma. Telomere lengths were measured in all specimens by restriction digestion of genomic DNA and Southern blot hybridization analysis. Associations between telomerase activity and tissue histopathology were assessed with two-sided Fisher's exact tests. RESULTS: Telomerase activity was detected in all examined germ cell cancers and in the benign testicular tissue specimens. In marked contrast, telomerase activity was not detected in any mature teratoma (P<.0001). Very long telomeres were detected in some mature teratomas, consistent with telomerase repression as a late event in teratoma formation. The immature teratoma, with malignant transformation, had high telomerase activity. CONCLUSION: Telomerase is active in germ cell cancers and repressed in mature teratomas. The absence of telomerase activity may contribute to the limited proliferative capacity of mature teratomas. These findings support the existence of an inverse relationship between telomerase activity and the differentiation state of clinical germ cell tumors.