Novel germ cell markers characterize testicular seminoma and fetal testis

Seminomas are characterized by expression of several stem cell markers, supporting their origin from germ cells. The current study focuses on novel germ cell markers in normal testes compared to those in fetal testes and different progression stages of seminomas. Microarray data were followed by RT-PCRs and immunohistochemistry on pure seminomas (pT1 to pT3) compared to adult and fetal testis. An upregulation of known germ cell markers, KIT, OCT4 and NANOG, was confirmed in seminoma specimens. We also identified novel germ cell markers such as BOB1 (POU2AF1, OBF1) and prominin 1 (PROM1, CD133), which were significantly upregulated in seminoma specimens, compared to normal testes. Furthermore, two Sertoli cell markers, SCGF (SCF) and the newly identified neuronal stem cell factor, MCFD2 (SDNSF), were expressed in seminoma cells. While BOB1 was expressed in fetal testis of second and third trimester of gestation, MCFD2 and PROM1 were only present in gonocytes up to the second trimester. All marker genes investigated were not further regulated in progressing tumour stages between pT1 and pT3. In conclusion, the germ cell markers described here provide evidence for the origin of seminoma cells, which could be from the developmental stage of early gonocytes or from spermatogonia re-expressing markers of the developing germ cells.     

The epigenome of testicular germ cell tumors

Gene expression is tightly regulated in normal cells, and epigenetic changes disturbing this regulation are a common mechanism in the development of cancer. Testicular germ cell tumor (TGCT) is the most common malignancy among young males and can be classified into two main histological subgroups: seminomas, which are basically devoid of DNA methylation, and nonseminomas, which in general have methylation levels comparable with other tumor tissues, as shown by restriction landmark genome scanning (RLGS). In general, DNA methylation seems to increase with differentiation, and among the nonseminomas, the pluripotent and undifferentiated embryonal carcinomas harbor the lowest levels of DNA promoter hypermethylation, whereas the well-differentiated teratomas display the highest. In this regard, TGCTs resemble the early embryogenesis. So far, only a limited number of tumor suppressor genes have been shown to be inactivated by DNA promoter hypermethylation in more than a minor percentage of TGCTs, including MGMT, SCGB3A1, RASSF1A, HIC1, and PRSS21. In addition, imprinting defects, DNA hypomethylation of testis/cancer associated genes, and the presence of unmethylated XIST are frequent in TGCTs. Aberrant DNA methylation has the potential to improve current diagnostics by noninvasive testing and might also serve as a prognostic marker for treatment response.     

Deletion of the short arm of chromosome 3 in breast tumors

Deletions in the short arm of chromosome 3 have long been known to be common in many tumor types, including carcinomas of the lung and kidney. Small interstitial deletions of the proximal-central region of 3p, with band 3p14 as a minimal common deleted segment, have recently been shown to occur in as many as 10% of carcinomas of the breast, often as the only chromosomal change. Seemingly identical deletions may also be found in the epithelial cells of mixed-lineage benign tumors of the breast and even in diffuse proliferative breast disease, a disorder that would not normally be accepted as neoplastic, but never in completely normal breast tissue. The cytogenetic evidence therefore indicates that the putative tumor suppressor gene deleted from 3p 14 influences cellular proliferation; evidently, its loss is often not sufficient for a fully malignant phenotype to emerge. The first information about FHIT, a candidate suppressor gene recently identified in the FRA3B fragile site in 3p14 and found to be abnormal or lost in a high percentage of carcinomas of various organs, including breast, is compatible with such a general proliferation-regulating role. Genes Chromosom. Cancer 18:241–245, 1997. © 1997 Wiley-Liss, Inc.     

Overrepresentation of chromosome 12p sequences and karyotypic evolution in i(12p)-negative testicular germ-cell tumors revealed by fluorescence in situ hybridization

Human testicular germ-cell tumors (TGCTs) comprise a heterogeneous group of solid neoplasms. These tumors are characterized by the presence of a highly specific chromosomal abnormality, i.e., an isochromosome of the short arm of chromosome 12. At present, this i(12p) chromosome is found in more than 80% of TGCTs. Isochromosome 12p has also been observed in some ovarian and extragonadal germ cell tumors. In the remaining so-called i(12p)-negative TGCTs other abnormalities involving chromosome 12, mainly 12p, can be found. In order to establish whether 12p abnormalities other than i(12p) are a common phenomenon in TGCTs, a panel of 11 i(12p)-negative tumors was investigated using multicolor fluorescence in situ hybridization. All TGCTs examined appeared to contain chromosomal abnormalities involving 12p, resulting in a distinct overrepresentation of short arm sequences. In addition, indications were obtained for a clonal evolution in one of the tumors. Our data suggest that the occurrence of 12p abnormalities is a common phenomenon in i(12p)-negative TGCTs and that these abnormalities, analogous to i(12p), may contribute to the process of tumor development.     

The association between intratubular seminoma and invasive germ cell tumors

Intratubular seminoma (ITS) has been defined as the complete filling of the seminiferous tubules with seminoma cells with no Sertoli cells present. This contrasts with intratubular germ cell neoplasia, unclassified (IGCNU), where the malignant germ cells are interspersed by Sertoli cells. We aimed to determine the relationship between these 2 entities and the association between ITS and invasive classic seminomas. We therefore examined the morphology and immunochemistry of ITS and IGCNU adjacent to germ cell tumors to differentiate the patterns, frequency, and distribution of these lesions. We found that ITS was seen in equal frequency adjacent to seminomas as it was to nonseminomas. The presence of ITS in non-seminomatous germ cell tumors suggests that it is a true in situ lesion rather than representative of intratubular spread of an existing seminoma. However, because it is not specifically associated with seminoma, we suggest that it is not useful to discriminate this lesion from IGCNU and that it merely represents an advanced form of IGCNU on the way to invasive malignancy.     

Identification of multiple chromosome 12 abnormalities in human testicular germ cell tumors by two-color fluorescence in situ hybridization (FISH)

The distribution of segments of the short and long arms of chromosome 12 was distinguished by two-color fluorescence in situ hybridization (FISH) in 27 cytogenetically abnormal testicular germ cell tumors (TGCTs). A 12p-specific probe was developed by chromosomal microdissection and sequence-independent polymerase chain reaction (PCR) amplification and was combined with a commercially available whole-chromosome 12 painting probe. The TGCTs included both i(12p)-positive and i(12p)-negative primary tumors and lymph node metastases from patients in clinical stage I or stage II who were not previously treated with chemotherapy. Rearrangements of the short arm of chromosome 12 and overrepresentation of 12p DNA sequences were found in all cases. In addition, cryptic rearrangements of 12p were found in 39% (7/18) of the i(12p)-positive tumors and in 78% (7/9) of the i(12p)-negative tumors. Only 7% (2/27) of all tumors had cryptic rearrangements of 12q.     

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.     

Nonrandom chromosome abnormalities in testicular and ovarian germ cell tumor cell lines

We report the karyotypic analysis of seven testicular and one ovarian germ cell tumor (GCT) cell lines, a number of which have previously been partially investigated. An i(12p) was found in each of the testicular GCT cell lines, while it was absent in the ovarian GCT cell line. Thus, our study extends to cell lines the observation from fresh tumor tissues that i(12p) is a highly nonrandom chromosomal abnormality of testicular GCT. Additional consistent nonrandom abnormalities in the testicular GCT cell lines included the following: del(1)(p22), del(1)(q21), i(1q), del(7)(q11.2), and del(12)(q14). The del(12)(q14) abnormality was identified in five of the cell lines investigated. This observation, together with previous detection of this marker chromosome in fresh tumor specimens by us and others, suggests that loss of genetic material on 12q may represent a primary change associated with malignant transformation of testicular germ cells. As reported in a previous study, a t(15;20)(p11;q11) translocation was identified in the ovarian GCT cell line. Interestingly, it also was seen in one testicular GCT cell line. In addition, a der(15)t(15;20)(p11;q11) marker chromosome was identified in two other testicular GCT cell lines. Thus, this reinvestigation of GCT cell lines has resolved the discrepancy regarding the occurrence of i(12p) in fresh tumors versus established cell lines and identified additional nonrandom abnormalities of potential importance to the development of GCTs.     

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.     

Fatty acid synthase overexpression in adult testicular germ cell tumors: potential role in the progression of non-seminomatous germ cell tumors

Overexpression of fatty acid synthase (FASN), which is a key enzyme responsible for the endogenous synthesis of fatty acids, and its association with multistep progression have been demonstrated in various human malignant tumors. We aimed to clarify the potential role of FASN overexpression in the development and progression of adult testicular germ cell tumors (TGCTs). From the primary sites of a cohort of 113 TGCT cases, we obtained 221 histological components: 53 intratubular germ cell neoplasias, unclassified (IGCNUs), 84 seminomas, 32 embryonal carcinomas, seven choriocarcinomas, 21 yolk sac tumors, and 24 teratomas. Samples were analyzed for overexpression of FASN by immunohistochemistry. Intensities of immunoreactivity and the fraction of positive cells were classified into each four categories (intensity, 0 to 3; fraction, 0–10 %?=?1, 11–50 %?=?2, 51–80 %?=?3, and >80 %?=?4). The overall score was determined by multiplication of both scores and overall scores greater than 6 were considered FASN overexpression. On a component basis, FASN overexpression was detected in 8 % of seminomas but not in IGCNUs (0 %) and was detected frequently in non-seminomatous germ cell tumors (NSGCTs) (88 % of embryonal carcinomas, all choriocarcinomas, 81 % of yolk sac tumors, and 54 % of teratomas). There were no cases of a mixed tumor (i.e., a tumor with multiple histological components) that overexpressed FASN in seminoma components but not in co-existing NSGCT components, suggesting sequential progression. Our immunohistochemical data suggest that FASN overexpression occurs as a late event during the progression from IGCNUs/seminomas to NSGCTs.     

A model for karyotypic evolution in testicular germ cell tumors

Testicular germ cell tumor karyotypes are characterized by near-triploidy, with chromosome numbers ranging from 50 to 70, and by the frequent appearance of i(12p). The high chromosome number has been attributed to the formation of tetraploid carcinoma in situ cells followed by chromosomal losses that ultimately lead to tumor forms that are more advanced. In the present investigation, we show by analysis of the accumulated cytogenetic data on testicular germ cell tumors and computer simulations that two distinct processes are operating in the karyotypic evolution of these tumors. The results suggest that whole-chromosome changes originate from a multipolar cell division of a tetraploid cell, whereas imbalances caused by structural changes accumulate in a stepwise manner.     

i(12p)-negative testicular germ cell tumors. A different group?

Cytogenetic analysis was performed of three seminomas, two primary nonseminomas, and two mature residual teratomas following chemotherapy, all lacking i(12p). Testicular germ cell tumors without an i(12p) may represent a subgroup of germ cell tumors, also in their clinical course, compared with those having i(12p).     

Abnormalities of the short arm of chromosome 12 in acute nonlymphocytic leukemia and dysmyelopoietic syndrome

Abnormalities of the short arm of chromosome #12 (12p) were found in 18 patients, 7 with previously untreated acute nonlymphocytic leukemia (ANLL) and 11 with dysmyelopoietic syndromes (MDS) or ANLL following treatment for another malignant disease. The chromosome #12 abnormality was a partial deletion in 15 patients and a translocation in 3. The 12p- was the sole chromosomal abnormality in seven patients (four with de novo ANLL) and was associated with other chromosome abnormalities in eight patients. Thus, partial monosomy for 12p was often associated with other chromosomal changes and was a secondary abnormality in some cases. The consequences of this hemizygosity for genes located at 12p are discussed with references to the possible expression of a potentially mutated recessive gene. The study of c-K-ras 2, normally located at 12p, must be done in such cases, as the association of secondary blood disorders and multiple chromosome abnormalities suggests a possible mutation of this c-oncogene on chromosome #12.     

Differential expression of RET finger protein in testicular germ cell tumors

Testicular germ cell cancer is a common cancer in young adults and its incidence has risen dramatically over the past several decades in Western countries. Because RET finger protein (RFP), which belongs to the large B-box RING finger protein family, has been reported to be expressed in different stages of spermatogenesis, we investigated its expression in testicular germ cell tumors. These comprised 13 pure seminomas, five pure non-seminomatous germ cell tumors (NSGCT) and seven mixed germ cell tumors, four of which contained seminomatous component. In normal adult testis, the expression of RFP was strong in the germ cells, particularly in spermatogonia and primary spermatocytes. RFP immunoreactivity was seen uniformly and specifically in 12 of the 13 pure seminomas examined. It was also detected in seminomatous components of mixed germ cell tumors, whereas pure NSGCT were negative for RFP expression. The expression of RFP in male germ cells and seminomas together with the lack of its expression observed in highly aggressive NSGCT suggested that RFP could be associated with the regulation of germ cell proliferation and/or histological-type of germ cell tumors.     

The role of spermatogenesis-associated protein 6 in testicular germ cell tumors

Objectives: To investigate the role of spermatogenesis-associated protein 6 (SPATA6) in the testicular germ cell tumors (TGCTs). Methods: Human embryonic carcinoma (EC)-derived cell line NTera2 was employed and randomly divided into normal control group, SPATA6c group, siSPATA6c group, and SPATA6c + siSPATA6c group. The recombinant expression vector pcDNA3.1 (+)-SPATA6 and target sequence for SPATA6-specific siRNA was transfected into NTera2 cells in the SPATA6c group and siSPATA6c group, respectively. The SPATA6 protein levels in each group were determined by Western blot. Cell proliferation and apoptosis rate were assessed by 3-(4, 5-dimethylthiazol-2-yl)-2 5-diphenyl-2Htetrazolium bromide (MTT) colorimetric assay and flow cytometry (FCM) assay, respectively. In addition, Western blot was performed to investigate the expression of Bax and B-cell lymphoma (Bcl)-2 in each group. Results: Compared with control group, protein levels of SPATA6 were significantly reduced in the siSPATA6c group, but were statistically increased in the SPATA6c group (P < 0.05). Similarly, the cell viability was significantly decreased by transfection with SPATA6 siRNA, but was increased by transfection with pcDNA3.1 (+)-SPATA6 compared with the control group. Moreover, the percentages of apoptosis cell were significantly higher in siSPATA6 group than those in the three groups. After transfection of SPATA6 siRNA, the expression of Bax was significantly increased, but the expression of Bcl-2 was markedly decreased than that in the control group and SPATA6c group. Conclusion: SPATA6 may play an important role in TGCTs, and down-regulation of SPATA6 could lead to apoptosis of TGCTs.     

Human testicular germ cell tumors in vitro and in athymic nude mice

Four cell lines derived from pure and mixed types of human testicular germ cell tumors in vitro and in nude mice were examined by light and electron microscopies. The NEC8 and NEC15 cell lines in vitro were composed of embryonal carcinoma cells with potentiality of trophoblastic differentiation. All of the tumors formed in nude mice by both cell lines were pure embryonal carcinomas. On the other hand, the NEC14 and ITO-II cell lines showed morphological differentiation from embryonal carcinoma cells to trophoblastic cells and to yolk sac tumor cells in vitro. In nude mice, these cell lines formed mixed tumors which consisted of embryonal carcinoma, yolk sac tumor, immature teratoma or trophoblastic cells. Our data suggest that some embryonal carcinomas have multipotentiality of morphological differentiation but that others have little such potentiality.     

The differential diagnosis of testicular germ cell tumors in theory and practice

Summary To better appreciate the conflicts and controversy surrounding the classification of testicular tumors, and to reappraise their morphologic substrate under the advent of tumor markers, 389 of our own cases are reviewed, classified according to the systems advocated by the World Health Organization (WHO) and the Testicular Tumour Panel and Registry (TTPR) of Great Britain, and evaluated statistically.While many cases fit easily into either classification, the following difficulties were manifest: 1) Discrepancies in definitions and diagnostic criteria are the reason that considerably more germ cell tumors could be classified as mixed choriocarcinomas (WHO) than as trophoblastic teratomas (TTPR). It was found that tumor markers supply histochemical data that often conflict with rather than supplement morphologic ones in diagnosis and differential diagnosis. Similarly, the incidence of yolk sac structures, as yet not recorded separately by the TTPR, varies as either morphologic or histochemical criteria are applied. 2) The division of the morphologic spectrum of teratomatous differentiation by criteria of distinction that are unequal in the two systems yield comparable but non-congruent tumor entities. Consequently, borderline cases may undergo shifts to noncorresponding groups as they are translated from one system to the other. 3) Criteria separating teratoma with malignant transformation and polyembryoma (WHO) from closely allied lesions proved impractical. 4) Diagnostic labels that incorporate not only a morphologic pattern but a definite level in the histogenetic hierarchy generate a climate of incompatibility between systems whose histogenetic perspectives differ. Embryonal carcinoma's claim to totipotence, in particular, leads to a conceptual split with the teratomas and brings the WHO system by itself into theoretic difficulties. Moreover, as the morphologic criteria for embryonal carcinoma are not in keeping with its histogenetic premise, the rigid separation is difficult to enforce in practice.Once the air is cleared, a resolution is easily reached. In the combined use of both classifications their real difference, splitting vs lumping, becomes a true asset.The cooperation of our documentation group PADOK is gratefully acknowledged