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.     

N- and KRAS mutations in primary testicular germ cell tumors: Incidence and possible biological implications

Recently, conflicting results have been reported on the incidence of RAS mutations in primary testicular germ cell tumors of adults (TGCTs). In four studies a low incidence of mutations (less than 15%) in a variety of TGCTs or derived cell lines was found, whereas in two other studies a high incidence of N- or KRAS mutations (over 40%) was shown. A total of 62 testicular seminomas (SE) and 34 nonseminomatous TGCTs (NS) were studied thus far. The largest series consisted of 42 TGCTs, studied on paraffin embedded tissue. We present the results of analysis for the presence of N- and KRAS mutations, in codons 12, 13, and 61, in snap frozen samples of 100 primary TGCTs, comprising 40 SE and 60 NS. Using the polymerase chain reaction (PCR) and allele specific oligonucleotide hybridization (ASO), mutations were found in five SE (three in NRAS and two in KRAS, all codon 12), and in one NS (KRAS, codon 12). To exclude underestimation of the incidence of RAS mutations in TGCTs due to the presence of an excess of wild type alleles in the analyzed sample, a PCR technique preferentially amplifying KRAS alleles with a mutation in codon 12 was applied to all SE. This approach, allowing a 250 times more sensitive assay, resulted in the detection of only one additional SE with a mutation. Based on a critical analysis of published data and on our results from the largest series of frozen samples investigated thus far, we conclude that N- or KRAS mutations are rare and apparently not essential for initiation or progression of TGCTs.     

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 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.     

Detection of RAS mutations in archival testicular germ cell tumors by polymerase chain reaction and oligonucleotide hybridization.

Preliminary studies of RAS mutational activation in human testicular germ cell neoplasms have yielded conflicting results. Whereas two studies of clinical material revealed a significant incidence of N- and KRAS mutations, two studies of a variety of germ cell lines failed to document RAS mutations. To clarify the incidence of RAS mutations in these tumors, we studied archival paraffin-embedded, formalin-fixed orchiectomy specimens from 25 nonseminomas (NSGCT), 18 seminomas (SEM), and one Leydig cell tumor. For 14 of the 44 neoplasms, DNA was also available from nonmalignant testis adjacent to the tumor. Six age-matched patients had testes removed because of nonmalignant disease and were studied as controls. Polymerase chain reaction (PCR) amplified the K-, N-, and HRAS 12, 13, and 61 codons of these specimens, and mutations were detected with mutation-specific oligonucleotide probe hybridization of Southern and slot blots. Four mutations were found in KRAS 12 (4/44;[9.1%]). One seminoma [1/18(5.6%)] contained the mutation GGT(GLY)----CGT(ARG), and three NSGCT [3/25(12%)] were found to have GGT(GLY)----GAT(ASP) mutations. One of the NSGCT mutations was detected in adjacent nonmalignant tissue, but the corresponding tumor did not contain any detectable mutation. No mutations were detected at KRAS 13 or 61, in NRAS or HRAS 12, 13, or 61, or in the control normal testes. PCR, slot blots, and hybridizations were performed twice by two separate investigators for confirmation of results. PCR-generated mutation-specific positive controls were created for all possible RAS mutations, and these along with wild-type DNA controls were integral to interpretation of the oligonucleotide mismatch hybridization assay. By using positive and negative controls, we have detected a relatively low incidence of RAS mutations in archival human testicular germ cell tumors.     

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.     

Analysis of the DND1 gene in men with sporadic and familial testicular germ cell tumors

A base substitution in the mouse Dnd1 gene resulting in a truncated Dnd protein has been shown to be responsible for germ cell loss and the development of testicular germ cell tumors (TGCT) in the 129 strain of mice. We investigated the human orthologue of this gene in 263 patients (165 with a family history of TGCT and 98 without) and found a rare heterozygous variant, p. Glu86Ala, in a single case. This variant was not present in control chromosomes (0/4,132). Analysis of the variant in an additional 842 index TGCT cases (269 with a family history of TGCT and 573 without) did not reveal any additional instances. The variant, p. Glu86Ala, is within a known functional domain of DND1 and is highly conserved through evolution. Although the variant may be a rare polymorphism, a change at such a highly conserved residue is characteristic of a disease-causing variant. Whether it is disease-causing or not, mutations in DND1 make, at most, a very small contribution to TGCT susceptibility in adults and adolescents.     

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.     

Molecular biology of testicular germ cell tumors: current status

Conclusions Changes in proto-oncogenes and tumor-suppressor genes at the molecular level are associated with the development and progression of testicular GCTs (Fig. 3). Investigations at this level, however, are only in their initial stages, and therefore the overall genetic changes which lead to the development of a metastasizing tumor are not known. Investigations show however, that undifferentiated GCTs (seminoma, embryonal carcinoma, chorionepithelioma) display molecular changes that are different from those of differentiated GCTs (teratocarcinoma, mixed tumors). In undifferentiated GCTs the following changes have been demonstrated: an increased expression of the proto-oncogenes c-kit, N-myc, c-myc, and c-mos; mutations in N-ras; missing expression in the RB tumor-suppressor gene; and a general hypomethylation of the DNA. These events possibly lead to a blockade of the differentiation process, and these GCTs may therefore correspond to an earlier stage of embryogenesis. These changes, on the other hand, do not occur in GCTs with differentiated tissue parts. The conspicuous expression of the c-erbB1 and c-erbB2 proto-oncogenes and also that of the RB tumor-suppressor gene is clearly associated in these tumors with differentiation. Important events in the formation or progression of teratocarcinoma and of the partly differentiated nonseminoma are, moreover, a generally lower number of copies of chromosome 15, a possible LOH at the nm23 locus, and hypermethylation, which may result in a switching off of particular genes.How the above molecular changes actually provide a clinically relevant supplement to the traditional classification of GCTs must be demonstrated by further investigations.Abbreviations GCT Germ cell turmor - CIS Carcinoma-in-situ - PCR Polymerase chain reaction - LOH Loss of heterozygosity - SCF Stem cell factor - SSCP Single Strand conformation polymorphism     

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.     

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     

Genetic control of susceptibility to spontaneous testicular germ cell tumors in mice

Testicular germ cell tumors (TGCTs) are the most common cancer affecting young men. TGCT is a polygenic trait and genes that control susceptibility for TGCT development have not yet been identified. The 129/Sv inbred strain of mice is an important experimental model to study the genetics and development of TGCTs. We review several novel approaches that were developed to study the susceptibility of TGCTs in the 129/Sv mouse model and its application in humans. These approaches showed that several spontaneous and engineered mutations interact with 129/Sv-derived susceptibility genes to enhance or suppress susceptibility; two of these mutations (Ter and Trp53) revealed novel linkages for susceptibility genes in sensitized polygenic trait analysis. Linkage analysis with a chromosome substitution strains suggests that as many as 100 genes control susceptibility. Bilateral TGCTs result from the coincidental occurrence of unilateral tumors. These results highlight the important contributions that this mouse model can make to studies of TGCT susceptibility in humans.     

Frequency of intratubular germ cell neoplasia with invasive testicular germ cell tumors. Histologic and immunocytochemical features

Intratubular germ cell neoplasia (ITGCN) has been regarded as the preinvasive stage of testicular germ cell tumors. We evaluated its frequency in 53 testicular germ cell tumors and determined whether immunohistochemical stains for alpha-fetoprotein, human chorionic gonadotropin, carcinoembryonic antigen, and ferritin demonstrated an advantage in its detection in comparison with hematoxylin-eosin and periodic acid-Schiff (PAS) stains. Residual seminiferous tubules were found at the periphery of the invasive neoplastic foci in 47 cases. Intratubular germ cell neoplasia was detected in several or multiple seminiferous tubules in 46 cases (98%). Exquisite localization of PAS-positive intracellular granules was present in all but one case of ITGCN. Focal immunocytochemical positivity for human chorionic gonadotropin, carcinoembryonic antigen, and ferritin was noted in 2.3% of cases. We conclude that ITGCN is present in most invasive germ cell tumors and the PAS stain is very reliable in its demonstration. Antigenic expression of the proteins that we examined is extremely limited in these primordial germ cells.     

Chromosomes, genes, and development of testicular germ cell tumors

A literature review found 265 articles on testicular germ cell tumors (TGCTs) detailing the copy number of chromosomal regions and expression of 245 genes. An initial precursor stage, intratubular germ cell neoplasia (IGCN), is characterized by triploidization and an upregulation of KIT, ALPP, CCDN2, and ZNF354A, and a downregulation of CDKN2D. TGCT regularly have a series of chromosomal aberrations: a decrease in copy number at 4q21 approximately qter and 5q14 approximately qter; an increase at 7p21 approximately pter, 7q21 approximately q33, and 8q12 approximately q23 (especially high increase in seminoma); a decrease at 11p11 approximately p15 and 11q14 approximately q24; an increase at 12p11 approximately pter; a decrease at 13q14 approximately q31; an increase of 17q11 approximately q21 (only for nonseminoma); a decrease of 18q12 approximately qter; and an increase at 21q21 approximately qter, 22q11 approximately qter (only for seminoma), and Xq. Macroscopically overt TGCT is associated with a characteristic series of abnormalities in the retinoblastoma pathway including upregulation of cyclin D2 and p27 and downregulation of RB1 and the cyclin-dependent kinase inhibitors p16, p18, p19, and p21. TGCT thus has a synergistic pattern in gene expressions of the retinoblastoma pathway that is rare in other malignancies.     

Pathogenesis of adult testicular germ cell tumors. A cytogenetic model

In essence, two models exist of the pathogenetic relationship between seminomas and nonseminomatous germ cell tumors (NSGCTs). In the first model, the histogenesis of seminomas is assumed to diverge from that of the other testicular germ cell tumors (TGCTs) at an early stage. The neoplastic pathway of seminomas and NSGCTs is different, with limited or no crossover. The second model suggests that seminomas and NSGCTs have a common origin with a single neoplastic pathway on which seminomas are an intermediate stage in development of NSGCTs. Our data on the cytogenetics and ploidy of seminomas, combined tumors, and NSGCTs lend support to the model of pathogenesis of seminomas and NSGCTs in which all TGCTs (with the possible exception of spermatocytic seminoma and infantile yolk sac tumor) have a single origin and neoplastic pathway, with seminomas representing an intermediate stage in development of NSGCT components, as opposed to the model in which seminomas and NSGCTs develop separately. The progression of TGCTs probably proceeds from high to lower numbers of chromosomes and is therefore accompanied by a net loss of chromosomal material. This decrease will be the end result of loss of specific chromosomes, gain of some other chromosomes (or part of chromosomes), and development of structural abnormalities.