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.     

Bilateral germ cell testicular tumors

PURPOSE: To study the clinical characteristics of bilateral testicular tumors in the cisplatin era. PATIENTS AND METHODS: Between November 1988 and November 1998 2386 testicular cancer patients were treated in our Department and 72 bilateral germ cell testicular cancer patients were retrospectively explored (3%). The incidence, the clinical and histological characteristics and, in the case of asynchronous tumor, the interval between the two tumors were analyzed. RESULTS: During the 10 years 19 synchronous (26.4%) and 53 asynchronous bilateral germ cell testicular cancers (73.6%) were treated. The incidence of bilateral synchronous seminoma was 68.4%. Among the asynchronous tumors 9 concordant seminomas and 9 concordant nonseminomas were detected. In the first, second and third 5-year follow-up period 39.6, 30.2, and 28.2% of asynchronous tumors were diagnosed. The incidence of seminoma after the first castration in the 5, 10 and 15 years was 19, 37.5, and 60%, respectively. The overall survival rates of synchronous and asynchronous testicular cancer were 84 and 93%. In cases of asynchronous tumor the prevalence of stage I cancer was significantly greater in a regularly controlled population (p=0.014) than in the not regularly followed population, but the survival rate was good in both groups. Nonseminoma showed up earlier as first and second tumor than seminoma (p=0.05, p=0.045). The interval between the two asynchronous tumors was shorter in the case of nonseminoma than in the case of seminoma (p=0.002). CONCLUSION: The prognosis of bilateral germ cell testicular cancer is good because of the high incidence rate of seminoma and the effective treatment. With regular follow-up the early diagnosis of second testicular tumors is probable. The interval between the tumors depends on the patients' age and the histology of the second tumor, in the case of seminoma it is longer. The effect of the previous treatment on the incidence of seminoma and the interval between the two asynchronous tumors requires further investigations.     

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.     

Serum AFP, hCG and CEA in the management of patients with testicular, ovarian and extragonadal germ cell tumors.

Serum levels of AFP, hCG and CEA were initially and serially measured in 59 patients with testicular germ cell tumors, and serially in 37 with ovarian and 3 with extragonadal germ cell tumors. Patients with seminoma/dysgerminoma or mature teratoma had normal serum AFP and sporadically slightly elevated hCG. Some patients with embryonal carcinoma, pure or with admixture of seminoma, had serum AFP elevated to maximum 100 U/ml, yet its use for monitoring therapy was limited. Patients with yolk sac tumors had elevated AFP and sometimes CEA levels, those with choriocarcinoma had elevated hCG, and those with compound tumors had one or more of the markers highly elevated. High AFP and/or hCG levels indicated the presence of the relevant tumor cells both in the primary and in residual tumor and/or metastases, also those missed in histological material, and thus were useful in restaging. Unfortunately, their absence in serum did not exclude the presence of marker-negative subpopulations of tumor cells. Changes in marker values paralleled the effects of treatment: the level increasing from any nadir heralded recurrence in patients in remission; elevated or increasing levels during therapy implied resistance to the therapy; decreasing levels indicated regression even though a return to the normal range did not mean eradication of all tumor cells.     

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.     

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 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.     

Immunocytochemical localization of oncodevelopmental proteins in human germ cell and hepatic tumors.

In a combined tissue and serum study alpha-1-antitrypsin (AAT) and alpha-fetoprotein are demonstrated in parallel within tumor tissue inclusions in both endodermal sinus (yolk sac) tumors and malignant hepatomas, and AAT is demonstrated as a marker in both neoplastic and preneoplastic liver lesions occurring in oral contraceptive users, all in association with normal serum AAT phenotype. The tumor inclusions in the first two instances differ immunocytochemically from AAT liver cell globules found in inherited AAT deficiency, which are unreactive for alpha-fetoprotein. It is concluded that unlike the molecular basis of storage associated with AAT phenotypic variation, the tumor inclusions reflect a separate, nongenetic mechanism of AAT storage, which may be epigenetic in nature. AAT and alpha-fetoprotein both are synthesized normally in yolk sac and fetal liver, a parallelism which disappears soon after birth. The reexpression of both proteins in two distinct tumor types arising from endodermal origins (yolk sac and liver), suggests that these markers may represent reemerging fetal gene products, a phenomenon previously proposed only for alpha-fetoprotein, a prototypic "oncofetal antigen."     

Ultrastructural localization and distribution of Nardilysin in mammalian male germ cells

Background

NRD convertase, also termed Nardilysin, is a Zn⁺⁺ metalloendopeptidase that specifically cleaves the N-terminus of arginine and lysine residues into dibasic moieties. Although this enzyme was found located within the testis, its function in male reproduction is largely unknown. In addition, the precise distribution of this enzyme within germ cells remains to be determined.

Methods

To answer these questions, we developed an immuno-gold electron microscopy analysis to detect Nardilysin at ultrastructural level in mice. In addition, we performed a quantitative analysis of these gold particles to statistically estimate the distribution of Nardilysin in the different subcellular compartments of differentiating late spermatids/spermatozoa.

Results

Expression of Nardilysin in wild-type mice was restricted to germ cells and markedly increased during the last steps of spermiogenesis. In elongated spermatids, we found the enzyme mainly localized in the cytoplasm, more precisely associated with two microtubular structures, the manchette and the axoneme. No labelling was detected over the membranous organelles of the spermatids. To test whether this localization is dependent of the functional microtubules organization of the flagella, we analysed the localization into a specific mouse mutant ebo/ebo (ébouriffé) known to be sterile due to an impairment of the final organization of the flagellum. In the ebo/ebo, the enzyme was still localized over the microtubules of the axoneme and over the isolated cytoplasmic microtubules doublets. Quantification of gold particles in wild-type and mutant flagella revealed the specific association of the enzyme within the microtubular area of the axoneme.

Conclusions

The strong and specific accumulation of Nardilysin in the manchette and axoneme suggests that the enzyme probably contributes either to the establishment of these specific microtubular structures and/or to their functional properties.

     

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.     

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.     

Serum and cerebrospinal fluid human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP) in intracranial germ cell tumors

We report a retrospective study on serum and cerebrospinal fluid (CSF) alpha-fetoprotein (AFP) and beta-human chorionic gonadotropin (betahCG) determination in a series of 30 patients bearing intracranial germ cell tumors. At diagnosis five patients had high serum and CSF AFP levels. No patient had positive serum AFP and negative CSF AFP or vice versa. Twelve of 30 patients had serum betahCG levels above 5 mlU/mL, eight had high betahCG only in CSF, and ten were completely negative. During treatment and follow-up both markers were accurate indicators of the response to therapy, decreasing rapidly and often becoming normal already after the first phase of treatment. We conclude that these two markers, and mostly betahCG, may be useful in the diagnosis and monitoring of the response to therapy of patients with intracranial germ cell tumors.     

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.     

Changes in testicular hCG binding and Leydig cell function in rats throughout life

The age dependence of Leydig cell function was investigated in rats from prepuberty (15 days) to senescence (39 months). Serum LH, serum and testicular testosterone were measured by radioimmunoassay. The binding capacity and affinity of LH/hCG receptors were determined by a radioligand receptor assay (hCG/Leydig cells) using 125I-hCG labelled by the lactoperoxidase method. Separation of bound and free 125I and simultaneous concentrations of 125I-hCG was achieved by vacuum ultrafiltration. The biochemical integrity of 125I-hCG tracer was ascertained by various chromatographic procedures. The highest hCG-finding and highest serum LH levels were found during puberty. Serum and testicular testosterone concentrations, however, were maximal in early adulthood. From this period onwards to late senescence hCG-binding changed only slightly, while serum LH and testosterone levels decreased significantly towards late senescence. The study shows that, although hCG binding to the Leydig cell changes characteristically during development, it is minimally affected by aging and cannot therefore be responsible for the reduced androgen biosynthesis in senescence.