Genetic aspects of testicular germ cell tumors

Testicular Germ Cell Tumor (TGCT) is the most common malignant tumor in young Caucasian men with an annual increase of 3-6% in the past 50 years. Data in the literature indicate that both environmental and genetic factors acting on the primordial gonocyte/gonocyte are implicated in the etiopathogenesis of this tumour. Genetic linkage and genome-wide analyses did not reveal a major gene effect so far, implying that multiple loci must contribute to the development of TGCTs. Only one significant genetic risk factor has been reported, the so called gr/gr deletion of the Y chromosome which still request further confirmation by independent studies. On the other side, the analysis of somatic genetic changes through mutation and genome imbalance analyses and expression profiling has just began to unravel the complex interaction of multiple pathways involved in TGCTs. This review focuses on genetic factors (both genomic and somatic) involved in the etiology, progression and treatment sensitivity of TGCTs.     

Pathobiology of testicular germ cell tumors: views and news

Human germ cell tumors (GCTs) are a heterogeneous group of neoplasms. They can occur in different anatomic locations, predominantly in the gonads (both ovary and testis) and in the midline of the body, including the retroperitoneal, mediastinal and hypothalamus/pineal gland regions. This distribution has been related to the migration routefollowed by primordial germ cells from the yolk sac to the genital ridge. The clinical behavior of these tumors depends on the sex of the patient, the age at clinical presentation and the histology of the tumor, Within the testis, three groups of GCTs can be distinguished; (I) yolk sac tumors and teratomas of neonates and infants; (II) seminomas and nonseminomas of adolescents and adults, the so-called testicular germ cell tumors; and (III) spermatocytic seminomas. This review discusses the histology, epidemiology and chromosomal constitution of GCT, in particular of the seminomas and nonseminomas of the adult testis, including their precursor, carcinoma in situ. In addition, the available data on the molecular basis of treatment sensitivity and resistance of GCT are reviewed.     

Effects of N-acetylcystein on bleomycin-induced apoptosis in malignant testicular germ cell tumors

Oxidative stress has been shown to induce apoptosis in cancer cells. Therefore, one might suspect that antioxidants may inhibit reactive oxygen species (ROS) and prevent apoptosis of cancer cells. No study has been carried out so far to elucidate the effects of N-acetylcysteine (NAC) on bleomycin-induced apoptosis in human testicular cancer (NCCIT) cells. We investigated the molecular mechanisms of apoptosis induced by bleomycin and the effect of NAC in NCCIT cells. We compared the effects of bleomycin on apoptosis with H₂O₂ which directly produces ROS. Strong antioxidant NAC was evaluated alone and in combination with bleomycin or H₂O₂ in germ cell tumor-derived NCCIT cell line (embryonal carcinoma, being the nonseminomatous stem cell component). We determined the cytotoxic effect of bleomycin and H₂O₂ on NCCIT cells and measured apoptosis markers such as caspase-3, caspase-8, and caspase-9 activities and Bcl-2, Bax, and cytochrome c (Cyt-c) levels in NCCIT cells incubated with bleomycin, H₂O₂, and/or NAC. We found half of the lethal dose (LD₅₀) of bleomycin on NCCIT cell viability as 120???g/ml after incubation for 72?h. Incubation with bleomycin (LD₅₀) induced increases in caspase-3, caspase-8, and caspase-9 activities and Cyt-c and Bax protein levels and a decrease in Bcl-2 level. Co-incubation of NCCIT cells with bleomycin and 10?mM NAC abolished bleomycin-induced increases in caspase-3 and caspase-9 activities, Bax, and Cyt-c levels and bleomycin-induced decrease in Bcl-2 level. Our results indicate that bleomycin induces apoptosis in NICCT cells and that NAC diminishes bleomycin-induced apoptosis via inhibiting the mitochondrial pathway. We conclude that NAC has negative effects on bleomycin-induced apoptosis in NICCT cells and causes resistance to apoptosis, which is not a desirable effect in the fight against cancer.     

Ultrastructural localization of hCG in the placenta and in testicular germ cell tumors

Human chorionic gonadotrophin(hCG)-producing cells in the human placenta and in testicular germ cell tumors have been investigated by ultrastructural immunohistochemistry using labeled Fab'fragments of anti-hCG-alpha and anti-hCG-beta. Most reaction products were demonstrated in the rough endoplasmic reticulum (rER) and perinuclear spaces of syncytiotrophoblastic giant cells ( STGC ) occurring in embryonal carcinomas and in syncytial cells observed in choriocarcinomas and in normal placenta. The rER of placental and choriocarcinomatous syncytial cells was highly developed and invariably showed a positive reaction. However, the rER of STGCs varied both with regard to its degree of development and its immunoreactivity with hCG. Localization of hCG in the Golgi apparatus and in cytoplasmic granules was indefinite. Mononuclear cells reacting positively with anti-hCG-alpha or -beta were also demonstrated. Cytotrophoblast-like cells which failed to react with anti-hCG-alpha or -beta were noted adjacent to sTGCs and the significance of these cells is discussed.     

A mini-review of familial ovarian germ cell tumors: An additional manifestation of the familial testicular germ cell tumor syndrome

Introduction While testicular germ cell tumors (TGCTs) are the most common malignancy in young men, germ cell tumors in women are uncommon. Familial clustering, epidemiologic evidence of increased risk with family or personal history of TGCT, and associations with genitourinary tract anomalies suggest an underlying genetic predisposition to TGCT, but traditional linkage studies have yet to identify a highly penetrant TGCT cancer susceptibility gene. In this paper, we investigate the familial occurrence of testicular and ovarian germ cell tumors. Methods We report a family in which a TGCT and an ovarian germ cell tumor (OGCT) occurred in two siblings, summarize the existing literature on familial occurrences of OGCT, either alone or in combination with extragonadal or TGCTs, and compare the incidence of familial and sporadic testicular and ovarian GCTs. Sporadic GCT data were obtained from the US Surveillance Epidemiology and End Results (SEER) registry. Results We identified 16 reports of OGCT occurring in conjunction with either ovarian, testicular or extragonadal GCT. In these familial cases, the mean age at onset of female dysgerminoma was younger than that noted in the general population (age 17 vs. age 24, p = 0.01). In SEER, the incidence of TGCT was 15 times higher than that of OGCT. Histologic distributions in males and females showed distinctly different patterns. Discussion Although the incidence of OGCTs in the general population is quite low, its occurrence in multiple members of the same family and in families with TGCT suggests that a gene conferring susceptibility to GCTs may exist in some families.     

Genetic variants in the 8q24 locus and risk of testicular germ cell tumors

Much evidence supports the premise that population genetic variation contributes significantly to the risk of testicular germ-cell tumor (TGCT). However, investigations of the association between genomic markers and TGCT susceptibility are scarce. Single nucleotide polymorphisms (SNPs) at the locus 8q24 have recently been found to be associated with prostate, colorectal and breast cancer. The US Servicemen’s testicular tumor environmental and endocrine determinants (STEED) study was used to investigate the association of 15 specific 8q24 SNPs with TGCT and its two main histologic groups of seminoma and nonseminoma. Conditional and unconditional logistic regression models, adjusted for the matching variables of year of birth, race/ethnicity and serum date, were utilized to produce odds ratios (OR) and 95% confidence intervals (95%CI). The analysis included 680 controls and 568 TGCT cases. In the TGCT analysis, no SNP was associated with risk in both heterozygotes and variant homozygotes. When stratified by histology the seminoma analysis also showed no association with the 8q24 SNPs. Conversely, the analysis of nonseminomas had three tentative associations (rs6470494, OR_{genotype AG} = 1.15, 95%CI: 0.86–1.54; OR_{genotype GG} = 1.68, 95%CI: 1.04–2.73; p for trend = 0.04) (rs13254738, OR_{genotype GT} = 1.04, 95%CI: 0.77–1.40; OR_{genotype TT} = 1.62, 95%CI: 1.06–2.49; p for trend = 0.07) (rs10505476, OR_{genotype CT} = 0.67, 95%CI: 0.50, 0.91; OR_{genotype TT} = 0.81, 95%CI: 0.47–1.39; p for trend = 0.04). There was no linkage disequilibrium between any of the 8q24 SNPs analyzed in this population. In conclusion, this study has found little evidence for an association between the reported 8q24 SNPs and TGCTs, although the findings for nonseminoma may be worth further investigation.     

A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumor-suppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.     

KIT (c-kit oncogene product) pathway is constitutively activated in human testicular germ cell tumors

We investigated the expression of KIT (product of c-kit oncogene), gain-of-function mutations, and activation of its downstream signal transduction in human testicular cancers. KIT was expressed in 88% (22/25) of seminomas and in 44.4% (4/9) of non-seminomas compared to adjacent normal testicular tissue. Nine of the KIT-expressing seminomas had mutations (40.9%; 9/22) in the c-kit gene; two cases in exon 11 and 7 cases in exon 17. Two of these mutations in exon 17 were novel, and the other seven mutations were identical to the already known gain-of-function mutations which cause activation of KIT without ligand stem cell factor. All of the mutant KIT and 53.8% (7/13) of wild-type KIT were phosphorylated (activated) and associated with phosphorylated phosphatidylinositol 3-kinase (PI3K). Akt was also phosphorylated in these seminomas, suggesting that the KIT-PI3K-Akt pathway is activated in seminoma. These findings suggest that the KIT-PI3K-Akt pathway is constitutively activated in testicular germ cell tumors, due to overexpression of KIT protein and/or gain-of-function mutations in the c-kit gene.     

Serum lactate dehydrogenase (S-LDH) and S-LDH isoenzymes in patients with testicular germ cell tumors

The activities of serum lactate dehydrogenase (S-LDH) and S-LDH isoenzymes were determined in 252 patients with a history of testicular germ cell tumors (TGCT). Fifteen of 37 patients with TGCT lesions and seven of 215 without had raised levels of S-LDH (above 8.0 mukat/l (480 U/l)). Of the patients with TGCT lesions, four had only raised S-LDH-1 levels, one only raised S-LDH-2 (and normal S-LDH), two only raised S-LDH-3 (one with normal S-LDH), and 10 had five combinations of raised levels of S-LDH isoenzymes with a predominance of S-LDH-1. S-LDH and S-LDH-1 correlated significantly with the total tumor volume in the patients with TGCT lesions, especially pronounced in those with lesions from seminoma. Of 34 patients with TGCT metastases, 13 with raised S-LDH levels lived significantly shorter lengths of time than 21 with normal S-LDH. Similarly, 11 with raised S-LDH-1 (above 3.0 mukat/l (180 U/l) lived significantly shorter times than 23 with normal S-LDH-1. S-LDH is a valuable tumor marker in patients with TGCT, especially in those with seminoma. Routine determination of S-LDH isoenzymes in addition to S-LDH in patients with TGCT is not recommended. In patients with a history of TGCT and an unexplained elevation of S-LDH levels, a raised S-LDH-1 level indicates the presence of TGCT lesions.     

Retroperitoneal and metachronous testicular germ cell tumors with different histology and teratoma growing syndrome--a case report

It is presented a case of a 32-year-old male with the three primary tumors diagnosed within a time period of 3 years; retroperitoneal nonseminoma in 2002, retroperitoneal mature teratoma in 2004, and metachronous testicular seminoma in 2005. We discuss the unusual presentation of these three rare events occurring in the same patient without known risk factors.     

Intermediate filament protein profiles of human testicular non-seminomatous germ cell tumors: correlation of cytokeratin synthesis to cell differentiation

The patterns of cytoskeletal differentiation were studied in 20 testicular non-seminomatous germ cell tumors by immunohistochemistry, using diverse monoclonal antibodies specific for different intermediate filament (IF) proteins and for desmoplakin. Immunofluorescence and immunoperoxidase methods on both formalin-fixed and frozen tissues were applied, in some cases together with a gel electrophoretic analysis of IF proteins. The tumors examined included embryonal carcinoma (EC), endodermal sinus tumor (EST), choriocarcinoma and teratoma. Nine of the tumors were composed of only one histological type, the others showed mixed components. Cytokeratins 8 and 18 were identified in all these neoplasms, but their immunostaining was weak in ECs. Cytokeratin 19 was absent or very scarce in ECs, but strongly expressed in ESTs, choriocarcinomas and teratomas, thus allowing the identification of small EST and choriocarcinoma elements in ECs even when they were morphologically not obvious. Occasionally, some cells in ECs and ESTs also stained for cytokeratins 4 and/or 17, indicating potential for epithelial stratification. The majority of the germ cell tumors showed varied amounts of vimentin, often in co-existence with cytokeratins. Neurofilaments were demonstrated in scattered tumor cells in a single case of EST. In the teratomas studied, each type of tissue component present showed the expected IF protein. However, in many germ cell tumors some stromal cells and blood vessels contained, in addition to vimentin and desmin, also cytokeratins 8 and 18. This heterogeneity of the cytoskeletal profile of germ cell tumors is indicative of the varied differentiation potential inherent in these neoplasms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular biomarkers as potential targets for therapeutic strategies in human testicular germ cell tumors: An overview

Testicular germ cell tumors (TGCTs), the most common malignancy in males between 15 and 34 years of age and the most frequent cause of death from solid tumors in this age group. TGCTs can be subdivided into seminoma and non‐seminoma germ cell tumors (NSGCTs), including embryonal cell carcinoma, choriocarcinoma, yolk sac tumor, and teratoma. Seminomas and NSGCTs do not only present distinctive clinical features, but they also show significant differences as far as therapy and prognosis are concerned. Seminomas are highly sensitive to both radiation and chemotherapy, with a good prognosis, non‐seminomas are sensitive to platinum‐based combination chemotherapy and are less susceptible to radiation, with the exception of teratomas. The different therapeutic outcome might be explained by inherent properties of the cells from which testicular neoplasia originate. The unique treatment sensitivity of TGCTs is unexplained so far, but it is likely to be related to intrinsic molecular characteristics of the PGCs/gonocytes, from which these tumors originate. Many discovered bio‐markers including OCT3/4, SOX2, SOX17, HMGA1, HMGA2, PATZ1, GPR30, Aurora B, estrogen receptor β, and others have given further advantages to discriminate between histological subgroups. In addition, therapeutic approaches for the treatment of TGCTs have been proposed: humanized antibodies against receptors/surface molecules on cancer cells, inhibitors of serine–threonine, and tyrosine kinases, and others. The mini‐review will be an overview on the molecular alterations identified in TGCTs and on novel targeted antineoplastic strategies that might help to treat chemotherapy resistant TGCTs. J. Cell. Physiol. 228: 1641–1646, 2013. © 2013 Wiley Periodicals, Inc.     

Aurora B expression in post-puberal testicular germ cell tumours

Aurora/Ipl1-related kinases are a conserved family of proteins that are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumours and have been implicated in mechanisms leading to mitotic spindle aberrations, aneuploidy, and genomic instability. Previous studies of our group have shown that Aurora B expression is restricted to specific germinal cells. In this study, we have evaluated by immunohistochemical analysis Aurora B expression in post-puberal testicular germ cell tumours (22 seminomas, 2 teratomas, 15 embryonal carcinomas, 5 mixed germinal tumours with a prominent yolk sac tumour component and 1 choriocarcinoma). The Aurora B protein expression was detected in all intratubular germ cell tumours, seminomas and embryonal carcinomas analysed but not in teratomas and yolk sac carcinomas. The immunohistochemical data were further confirmed by Western blot analysis. In addition, the kinase Aurora B was vigorously expressed in GC-1 cells line derived from murine spermatogonia. The block of Aurora B function induced by a pharmacological inhibitor significantly reduced the growth of GC-1 cells suggesting that Aurora B is a potential therapeutic target. J. Cell. Physiol. 221: 435–439, 2009. © 2009 Wiley-Liss, Inc.     

Differentiation capabilities of human germ cell tumors

It is well known that human germ cell tumors are an excellent model to study not only differentiation capacity of tumor cells but also human normal somatic cell differentiation. A variety of polyclonal and monoclonal antibodies were developed against cell surface antigens of murine embryos and teratocarcinomas. Accumulated data has revealed that these antigens are sequentially expressed on embryonic cells in a well-programmed manner. They have also been shown to be useful markers to investigate somatic cell differentiation in fetal and adult tissue. In humans, however, little is known about the cellular differentiation mechanism in early embryos and whether they could be studied, i.e. whether they occur in human germ cell tumors. In present review, we discussed newly established monoclonal antibodies which were raised from human embryonal carcinoma cells. We have been studying differentiation capacity of human germ cell tumor cells by using these antibodies. Some of these antibodies clearly indicates their usefulness to specify the developmental stage of normal tissue.     

The effects of steel mutation on testicular germ cell differentiation

The effects of artificial cryptorchidism and its surgical reversal on spermatogenesis were examined in germ cell mutant, S1/+ and wild type, +/+, mice. In cryptorchid testes no difference was found between S1/+ and +/+ mice in the number of undifferentiated type A spermatogonia. The activity of type A spermatogonia in mutant mice appeared normal as judged by its mitotic cell number and DNA synthesis. The surgical reversal of cryptorchidism resulted in regenerative differentiation of mature germ cells in both types of mice, but the pattern of cellular differentiation in the mutant testes was completely different from that of the wild type testes. At two steps of cellular differentiation, intermediate or type B spermatogonia and spermatid, the numbers of cells were much smaller in the S1/+ testes than those in the +/+ testes. The steel gene was therefore suggested to exert its effects on the differentiation of type A spermatogonia to intermediate or type B spermatogonia, on meiotic division and/or the survival rate of these cells, but not on the undifferentiated type A spermatogonia or stem cells.     

Functional role of caspases in heat-induced testicular germ cell apoptosis

In the present study, we determined whether a pan caspase inhibitor could prevent or attenuate heat-induced germ cell apoptosis. Groups of five adult (8 wk old) C57BL/6 mice pretreated with vehicle (DMSO) or Quinoline-Val-Asp (Ome)-CH2-O-Ph (Q-VD-OPH), a new generation broad-spectrum caspase inhibitor, were exposed once to local testicular heating (43 degrees C for 15 min) and killed 6 h later. The inhibitor (40 mg/kg body weight) or vehicle was administered intraperitoneally (i.p.) 1 h before local testicular heating. Germ cell apoptosis was detected by TUNEL assay and quantitated as number of apoptotic germ cells per 100 Sertoli cells at stages XI-XII. Compared with controls (16.8 +/- 3.1), mild testicular hyperthermia within 6 h resulted in a marked activation (277.3 +/- 21.6) of germ cell apoptosis, as previously reported by us. Q-VD-OPH at this dose markedly inhibited caspase 3 activation and significantly prevented (by 67.0%) heat-induced germ cell apoptosis. Q-VD-OPH-mediated rescue of germ cells was independent of cytosolic translocation of mitochondrial cytochrome c and DIABLO. Electron microscopy further revealed normal appearance of these rescued cells. Similar protection from heat-induced germ cell apoptosis was also noted after pretreatment with minocycline, a second-generation tetracycline that effectively inhibits cytochrome c release and, in turn, caspase activation. Collectively, the present study emphasizes the role of caspases in heat-induced germ cell apoptosis.