Diagnostic Capability of Cerebrospinal Fluid-Placental Alkaline Phosphatase Value in Intracranial Germ Cell Tumor.

OBJECTIVE: Most types of intracranial germ cell tumors (IGCTs) are sensitive to chemoradiation. However, biopsy specimens are usually small and thus cannot be used for obtaining an accurate pathological diagnosis. Recently, the cerebrospinal fluid (CSF) placental alkaline phosphatase (PLAP) value has been considered a new biomarker of IGCTs. The present study aimed to evaluate the discriminatory characteristics of the CSF-PLAP value upon diagnosis and at the time of recurrence in patients with IGCTs. METHODS: Between 2015 and 2019, this study included 37 patients with tumors located in the intraventricular and/or periventricular region. The CSF-PLAP level was assessed before the patients received any treatment. The PLAP level was evaluated during and after first-line chemoradiotherapy in 7 patients with IGCTs. The CSF-PLAP values were compared according to histological diagnosis, and the correlation between these values and radiographical features was assessed. The CSF-PLAP values of 6 patients with IGCTs with suspected recurrence were evaluated based on neuroimaging findings. RESULTS: The CSF-PLAP values were significantly higher in patients with IGCTs than in those with other types of brain tumor (n = 19 vs. 18; median: 359.0 vs. <8.0 pg/mL). The specificity and sensitivity were 88 and 95%, respectively, with a cutoff value of 8.0 pg/mL. In patients with IGCT, the CSF-PLAP value was higher in patients with germinoma than in those with nongerminomatous germ cell tumors (n = 12 vs. 7; median: 415.0 vs. 359.0 pg/mL). Regarding the time course, the CSF-PLAP value decreased to below the detection limit after the reception of first-line chemoradiotherapy in all 7 patients. A significant correlation was observed between the initial CSF-PLAP value and the tumor reduction volume after receiving first-line chemoradiotherapy (p < 0.0003, R2 = 0.6165, logY = 1.202logX - 1.727). Among the patients with suspected IGCT recurrence (n = 6), the CSF-PLAP value was high in patients with recurrence (n = 3; median: 259.0 pg/mL), and that in patients (n = 3) without recurrence was below the lower detection limit. CONCLUSIONS: The CSF-PLAP level is a useful biomarker during the initial diagnosis of IGCTs and at the time of recurrence. It may be associated with the volume of germinomatous components of tumors.

Differential expression of DNA methyltransferases and demethylases among the various testicular germ cell tumor subtypes.

Aim: Characterize DNA methyltransferases/demethylases expression in testicular germ cell tumors (TGCTs). Methods:In silico analysis of TCGA database, assessment of transcript levels of most relevant enzymes in four TGCT cell lines and validation in patient cohort (real-time quantitative polymerase chain reaction; immunohistochemistry). Results:DNMT3A, DNMT3B and TET2 were the most differentially expressed between seminomas (SEs) and nonseminomas (NSs). DNMT3B was significantly overexpressed in NS-related cell lines, and the opposite was found for TET2. Significantly higher DNMT3A/B mRNA expression was observed in NS, indicating a role for de novo methylation in reprogramming. Significantly higher TET2 protein expression was observed in SEs, suggesting active demethylation contributes for SE hypomethylated state. More differentiated histologies disclosed distinct expression patterns. Conclusion: DNA-modifying enzymes are differentially expressed between TGCT subtypes, influencing reprogramming and differentiation.

TdT expression in germ cell tumours: a possible immunohistochemical cross-reaction and diagnostic pitfall.

AIMS: Very recent papers proposed a possible role for the expression of terminal deoxynucleotidyl transferase (TdT) in the tumourigenesis of gonadal and extragonadal germ cell-derived tumours (GCTs). Our multicentric study evaluated the magnitude of the immunoreactivity for TdT in GCTs, encompassing seminoma, dysgerminoma, mature teratoma and mixed GCTs. METHODS AND RESULTS: The histological series was stained with both monoclonal and polyclonal antibodies, yielding a positivity of 80% of cases with well-defined nuclear reactivity. A significant difference in staining intensity between monoclonal and polyclonal antibodies was observed (p=0.005). However, exploiting western blot and more innovative proteomic approaches, no clear-cut evidence of the TdT protein was observed in the neoplastic tissues of the series. CONCLUSIONS: Alternatively to the pathogenetic link between TdT expression and GCTs tumourigenesis, we hypothesised the occurrence of a spurious immunohistochemical nuclear cross-reaction, a well-known phenomenon with important implications and a possible source of diagnostic pitfalls in routine practice for pathologists.

Vitamin C Attenuates Sodium Fluoride-Induced Mitochondrial Oxidative Stress and Apoptosis via Sirt1-SOD2 Pathway in F9 Cells.

Increasing evidence has suggested an important role played by reactive oxygen species (ROS) in the pathogenesis of fluorosis. Accumulating evidence demonstrates that vitamin C administration ameliorate sodium fluoride (NaF)-induced oxidative stress. However, the potentially beneficial effects of vitamin C against NaF-induced cytotoxicity and the underlying molecular mechanisms of this protection are not fully understood. Here, we found that NaF stimulated cytotoxicity, increased mitochondrial reactive oxygen species (mROS) production, and induced apoptosis in F9 embryonic carcinoma cells. Consistent with this finding, NaF exposure was associated with decreased Sirtuin 1 (Sirt1) protein expression, thus promoted the acetylation of manganese superoxide dismutase (SOD2), a key enzyme involved in regulating mROS production. However, all NaF-induced mitochondrial oxidative injuries were efficiently ameliorated by overexpression of Sirt1 or incubation with Mito-TEMPO (a SOD2 mimetic). Moreover, pretreatment with vitamin C enhanced the expression of Sirt1 and decreased NaF-induced mitochondrial oxidative stress and apoptosis. Knockdown of Sirt1 blocked the vitamin C-mediated reduction in mROS and apoptosis via inhibiting Sirt1-SOD2 signaling. Importantly, sodium-dependent vitamin C transporter 2 (SVCT-2) siRNA was found to partially block the ability of vitamin C to promote Sirt1/SOD2 signaling. In summary, our data indicate that Sirt1 plays a pivotal role in the ability of vitamin C to stimulate SOD2 activity and attenuate mitochondrial oxidative stress, which partially through vitamin C receptor in NaF-induced F9 cells injury.

Germ cell neoplasia in situ complicating 17ß-hydroxysteroid dehydrogenase type 3 deficiency.

17ß-hydroxysteroid dehydrogenase type 3 (17ßHSD3) deficiency is an autosomal recessive disorder of male sex development that results in defective testosterone biosynthesis. Although mutations in the cognate HSD17B3 gene cause a spectrum of phenotypic manifestations, the majority of affected patients are genetic males having female external genitalia. Many cases do not present until puberty, at which time peripheral conversion of androgen precursors causes progressive virilization. Measurement of the testosterone-to-androstenedione ratio is useful to screen for 17ßHSD3 deficiency, and genetic analysis can confirm the diagnosis. As some individuals with 17ßHSD3 deficiency transition from a female sex assignment to identifying as males, providers should ensure stable gender identity prior to recommending irreversible treatments. Gonadectomy is indicated to prevent further virilization if a female gender identity is established. The risk of testicular neoplasia is unknown, a point which should be discussed if patients elect to transition into a male gender role.

Expression patterns of HENMT1 and PIWIL1 in human testis: implications for transposon expression.

This study aimed to define the expression patterns of HENMT1 and PIWI proteins in human testis and investigate their association with transposon expression, infertility sub-type or development of testicular germ cell tumours (TGCTs). Testis biopsies showing normal spermatogenesis were used to identify normal localisation patterns of HENMT1 and PIWIL1 by immunolocalisation and RT-PCR after laser microdissection. 222 testis biopsies representing normal spermatogenesis, hypospermatogenesis, spermatogenic arrests, Sertoli cell-only (SCO) tumours and TGCTs were analysed by RT-qPCR for expression of HENMT1/PIWIL1/PIWIL2/PIWIL3/PIWIL4 and LINE-1 Additionally, HENMT1-overexpressing TCam2 seminoma cell lines were analysed for the same parameters by RT-qPCR. We found that HENMT1 and PIWIL1 are coexpressed in pachytene spermatocytes and spermatids. Expression of HENMT1, PIWIL1 and PIWIL2 was mainly dependent on germ cell content but low levels of expression were also detected in some SCO samples. Levels of HENMT1, PIWIL1 and PIWIL2 expression were low in TGCT. Samples with HENMT1, PIWIL2 and PIWIL4 expression showed significantly (P < 0.05) lower transposon expression compared to samples without expression in the same histological group. HENMT1-overexpressing TCam2 cells showed lower LINE-1 expression than empty vector-transfected control lines. Our findings support that the transposon-regulating function of the piRNA pathway found in the mouse is conserved in adult human testis. HENMT1 and PIWI proteins are expressed in a germ-cell-specific manner and required for transposon control.

An Overview on Predictive Biomarkers of Testicular Germ Cell Tumors.

Testicular germ cell tumors (TGCTs) are frequent solid malignant tumors and cause of death in men between 20-40 years of age. Genetic and environmental factors play an important role in the origin and development of TGCTs. Although the majority of TGCTs are responsive to chemotherapy, about 20% of patient presents incomplete response or tumors relapse. In addition, the current treatments cause acute toxicity and several chronic collateral effects, including sterility. The present mini-review collectively summarize the most recent findings on the new discovered molecular biomarkers such as tyrosine kinases, HMGAs, Aurora B kinase, and GPR30 receptor predictive of TGCTs and as emerging new possible molecular targets for therapeutic strategies. J. Cell. Physiol. 232: 276-280, 2017. © 2016 Wiley Periodicals, Inc.

Wilms tumor gene 1 (WT1), TP53, RAS/BRAF and KIT aberrations in testicular germ cell tumors.

PURPOSE: Wilms tumor gene 1 (WT1), a zinc-finger transcription factor essential for testis development and function, along with other genes, was investigated for their role in the pathogenesis of testicular germ cell tumors (TGCT). METHODS: In total, 284 TGCT and 100 control samples were investigated, including qPCR for WT1 expression and BRAF mutation, p53 immunohistochemistry detection, and massively parallel amplicon sequencing. RESULTS: WT1 was significantly (p < 0.0001) under-expressed in TGCT, with an increased ratio of exon 5-lacking isoforms, reaching low levels in chemo-naïve relapsed TGCT patients vs. high levels in chemotherapy-pretreated relapsed patients. BRAF V600E mutation was identified in 1% of patients only. p53 protein was lowly expressed in TGCT metastases compared to the matched primary tumors. Of 9 selected TGCT-linked genes, RAS/BRAF and WT1 mutations were frequent while significant TP53 and KIT variants were not detected (p = 0.0003). CONCLUSIONS: WT1 has been identified as a novel factor involved in TGCT pathogenesis, with a potential prognostic impact. Distinct biologic nature of the two types of relapses occurring in TGCT has been demonstrated. Differential mutation rate of the key TGCT-related genes has been documented.

DNA methyltransferase-3 like protein expression in various histological types of testicular germ cell tumor.

OBJECTIVE: DNA methyltransferase 3-like plays an important role in germ cell development. The aim of this study was to analyse the DNA methyltransferase 3-like protein expression in testicular germ cell tumors. METHODS: The immunohistochemical expression of DNA methyltransferase 3-like was examined in 86 testicular germ cell tumor specimens in various clinical settings. The association between DNA methyltransferase 3-like expression and disease stage was analyzed. RESULTS: DNA methyltransferase 3-like was strongly expressed in seven of the eight pure embryonal carcinomas (87.5%). Partial DNA methyltransferase 3-like expression was observed in 6 of 23 (26.1%) pure seminomas. Various degrees of DNA methyltransferase 3-like expression was observed in all four pure yolk sac tumors, of which three were prepubertal yolk sac tumors. In mixed germ cell tumors, DNA methyltransferase 3-like protein was expressed in various degrees in elements of the embryonal carcinoma (14/18, 77.8%), seminoma (4/11, 36.4%), teratoma (4/7, 57.1%) and choriocarcinoma (3/3, 100%) but not in the yolk sac tumors (0/4). When DNA methyltransferase 3-like expression was analyzed according to disease stages, it was significantly correlated with advanced seminoma rather than Stage I seminoma (46.2 vs. 0%, P = 0.019), whereas there was no significant difference in the DNA methyltransferase 3-like-positive proportion between Stage I and advanced disease in the mixed germ cell tumors. CONCLUSIONS: Our findings suggest that DNA methyltransferase 3-like protein may play roles not only in the development of embryonal carcinoma but also in the development of advanced pure seminoma and pure yolk sac tumor.

Rare inactivating PDE11A variants associated with testicular germ cell tumors.

Germline inactivating mutations of isoform 4 of phosphodiesterase (PDE) 11A (coded by the PDE11A gene) have been associated with familial adrenocortical tumors and familial testicular cancer. Testicular tissue is unique in expressing all four isoforms of PDE11A. In a prior candidate gene study of 94 familial testicular germ cell tumor (TGCT) subjects, we identified a significant association between the presence of functionally abnormal variants in PDE11A and familial TGCT risk. To validate this novel observation, we sequenced the PDE11A coding region in 259 additional TGCT patients (both familial and sporadic) and 363 controls. We identified 55 PDE11A variants: 20 missense, four splice-site, two nonsense, seven synonymous, and 22 intronic. Ten missense variants were novel; nine occurred in transcript variant 4 and one in transcript variant 3. Five rare mutations (p.F258Y, p.G291R, p.V820M, p.R545X, and p.K568R) were present only in cases and were significantly more common in cases vs controls (P=0.0037). The latter two novel variants were functionally characterized and shown to be functionally inactivating, resulting in reduced PDE activity and increased cAMP levels. In further analysis of this cohort, we focused on white participants only to minimize confounding due to population stratification. This study builds upon our prior reports implicating PDE11A variants in familial TGCT, provides the first independent validation of those findings, extends that work to sporadic testicular cancer, demonstrates that these variants are uncommonly but reproducibly associated with TGCT, and refines our understanding regarding which specific inactivating PDE11A variants are most likely to be associated with TGCT risk.

Serum metallothionein in patients with testicular cancer.

PURPOSE: Metallothioneins (MTs) have been disclosed as a useful diagnostic factor for tumour progression and drug resistance in a variety of malignancies. Increased levels of MT in blood serum have been found in patients with several types of cancer, but there is no available information on serum MT levels in patients with testicular germ cell tumour (TGCT). The aim of the study was to determine MT levels in serum of patients with TGCT and to evaluate the portion of platinum (Pt) that binds to MT after cisplatin administration since MTs could be involved in drug resistance. METHODS: Concentration of total MT was determined in serum of 25 men with newly diagnosed TGCT by differential pulse voltammetry. The fractionation of serum was carried out by size exclusion high-performance liquid chromatography (SE-HPLC), while concentration of Pt in collected fractions was determined by inductively coupled plasma mass spectrometry. RESULTS: Concentration of serum MT was significantly higher in TGCT patients than in healthy volunteers. The results of SE-HPLC analysis showed that only a small amount of Pt was bound to proteins in the area of MT elution. CONCLUSIONS: Significant increase in MT levels in individuals with TGCT indicates certain health problem and, in combination with other commonly used diagnostic tools, could improve early diagnosis.

EMMPRIN/CD147-encriched membrane vesicles released from malignant human testicular germ cells increase MMP production through tumor-stroma interaction.

BACKGROUND: Elevated levels of EMMPRIN/CD147 in cancer tissues have been correlated with tumor progression but the regulation of its expression is not yet understood. Here, the regulation of EMMPRIN expression was investigated in testicular germ cell tumor (TGCTs) cell lines. METHODS: EMMPRIN expression in seminoma JKT-1 and embryonal carcinoma NT2/D1 cell lines was determined by Western blot, immunofluorescence and qRT-PCR. Membrane vesicles (MVs) secreted from these cells, treated or not with EMMPRIN siRNA, were isolated by differential centrifugations of their conditioned medium. MMP-2 was analyzed by zymography and qRT-PCR. RESULTS: The more aggressive embryonic carcinoma NT2/D1 cells expressed more EMMPRIN mRNA than the seminoma JKT-1 cells, but surprisingly contained less EMMPRIN protein, as determined by immunoblotting and immunostaining. The protein/mRNA discrepancy was not due to accelerated protein degradation in NT2/D1 cells, but by the secretion of EMMPRIN within MVs, as the vesicles released from NT2/D1 contained considerably more EMMPRIN than those released from JKT-1. EMMPRIN-containing MVs obtained from NT2/D1, but not from EMMPRIN-siRNA treated NT2/D1, increased MMP-2 production in fibroblasts to a greater extent than those from JKT-1 cells. CONCLUSION AND GENERAL SIGNIFICANCE: The data presented show that the more aggressive embryonic carcinoma cells synthesize more EMMPRIN than seminoma cells, but which they preferentially target to secreted MVs, unlike seminoma cells which retain EMMPRIN within the cell membrane. This cellular event points to a mechanism by which EMMPRIN expressed by malignant testicular cells can exert its MMP inducing effect on distant cells within the tumor microenvironment to promote tumor invasion. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Identification of ZDHHC14 as a novel human tumour suppressor gene.

Genomic changes affecting tumour suppressor genes are fundamental to cancer. We applied SNP array analysis to a panel of testicular germ cell tumours to search for novel tumour suppressor genes and identified a frequent small deletion on 6q25.3 affecting just one gene, ZDHHC14. The expression of ZDHHC14, a putative protein palmitoyltransferase with unknown cellular function, was decreased at both RNA and protein levels in testicular germ cell tumours. ZDHHC14 expression was also significantly decreased in a panel of prostate cancer samples and cell lines. In addition to our findings of genetic and protein expression changes in clinical samples, inducible overexpression of ZDHHC14 led to reduced cell viability and increased apoptosis through the classic caspase-dependent apoptotic pathway and heterozygous knockout of ZDHHC14 increased [CORRECTED] cell colony formation ability. Finally, we confirmed our in vitro findings of the tumour suppressor role of ZDHHC14 in a mouse xenograft model, showing that overexpression of ZDHHC14 inhibits tumourigenesis. Thus, we have identified a novel tumour suppressor gene that is commonly down-regulated in testicular germ cell tumours and prostate cancer, as well as given insight into the cellular functional role of ZDHHC14, a potential protein palmitoyltransferase that may play a key protective role in cancer.

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.

Tumor-suppressive function of protein-tyrosine phosphatase non-receptor type 23 in testicular germ cell tumors is lost upon overexpression of miR142-3p microRNA.

Protein-tyrosine phosphatase non-receptor type 23 (PTPN23) is a candidate tumor suppressor involved in the tumorigenesis of various organs. However, its physiological role(s) and detailed expression profile(s) have not yet been elucidated. We investigated the function and regulation of PTPN23 in the formation of testicular germ cell tumors (TGCTs). Expression of PTPN23 in human TGCT cell lines was significantly lower than that in spermatogonial stem cells in mice. Overexpression of PTPN23 in NEC8, a human TGCT cell line, suppressed soft agar colony formation in vitro and tumor formation in nude mice in vivo. These data indicate that PTPN23 functions as a tumor suppressor in TGCTs. Multiple computational algorithms predicted that the 3' UTR of human PTPN23 is a target for miR-142-3p. A luciferase reporter assay confirmed that miR-142-3p bound directly to the 3' UTR of PTPN23. Introduction of pre-miR-142 in the PTPN23 transfectant of NEC8 led to suppressed expression of PTPN23 and increased soft agar colony formation. Quantitative RT-PCR data revealed a significantly higher expression of miR-142-3p in human seminomas compared with normal testes. No difference in mRNA expression between seminoma and non-seminoma samples was detected by in situ hybridization. Both quantitative RT-PCR and immunohistochemical analyses revealed that PTPN23 expression was significantly lower in TGCTs than in normal testicular tissues. Finally, a lack of PTPN23 protein expression in human TGCTs correlated with a relatively higher miR-142-3p expression. These data suggest that PTPN23 is a tumor suppressor and that repression of PTPN23 expression by miR-142-3p plays an important role in the pathogenesis of TGCTs.

PARP expression in germ cell tumours.

BACKGROUND: Poly(ADP-ribose)polymerase (PARP) inhibitors represent a new class of promising drugs in anticancer therapy. AIMS: To evaluate PARP expression in testicular germ cell tumours (GCTs) and to correlate expression patterns with clinicopathological variables. METHODS: In this translational study, tumour specimens from 124 patients with GCTs (114 patients with testicular primary tumours and 10 with extragonadal GCTs) were identified. PARP expression was detected by immunohistochemistry using monoclonal antibodies, scored by the multiplicative quickscore (QS) method and compared to PARP expression in normal testicular tissue. RESULTS: We observed higher expression of PARP in testicular tumours compared to normal testicular tissue (mean QS=10.04 vs 3.31, p<0.0000001). Mean QS±SD for each histological subtype was as follows: intratubular germ cell neoplasia unclassified (IGCNU)=18.00±0.00, embryonal carcinoma=9.62±5.64, seminoma=9.74±6.51, yolk sac tumour=7.8±7.20, teratoma=5.87±5.34, and choriocarcinoma=4.50±8.33. The PARP overexpression (QS>9) was most often detected in IGCNU (100% of specimen with PARP overexpression), seminona (52.6%), embryonal carcinoma (47.0%), yolk sac tumour (33.3%), teratoma (26.7%) and choriocarcinoma (25.0%), compared to 1.9% of normal testicular tissue specimens. There was no association between PARP expression and clinical variables. CONCLUSIONS: In this pilot study, we showed for the first time, that PARP is overexpressed in testicular germ cell tumours compared to normal testis.

Reduced proficiency in homologous recombination underlies the high sensitivity of embryonal carcinoma testicular germ cell tumors to Cisplatin and poly (adp-ribose) polymerase inhibition.

Testicular Germ Cell Tumors (TGCT) and patient-derived cell lines are extremely sensitive to cisplatin and other interstrand cross-link (ICL) inducing agents. Nevertheless, a subset of TGCTs are either innately resistant or acquire resistance to cisplatin during treatment. Understanding the mechanisms underlying TGCT sensitivity/resistance to cisplatin as well as the identification of novel strategies to target cisplatin-resistant TGCTs have major clinical implications. Herein, we have examined the proficiency of five embryonal carcinoma (EC) cell lines to repair cisplatin-induced ICLs. Using γH2AX staining as a marker of double strand break formation, we found that EC cell lines were either incapable of or had a reduced ability to repair ICL-induced damage. The defect correlated with reduced Homologous Recombination (HR) repair, as demonstrated by the reduction of RAD51 foci formation and by direct evaluation of HR efficiency using a GFP-reporter substrate. HR-defective tumors cells are known to be sensitive to the treatment with poly(ADP-ribose) polymerase (PARP) inhibitor. In line with this observation, we found that EC cell lines were also sensitive to PARP inhibitor monotherapy. The magnitude of sensitivity correlated with HR-repair reduced proficiency and with the expression levels and activity of PARP1 protein. In addition, we found that PARP inhibition strongly enhanced the response of the most resistant EC cells to cisplatin, by reducing their ability to overcome the damage. These results point to a reduced proficiency of HR repair as a source of sensitivity of ECs to ICL-inducing agents and PARP inhibitor monotherapy, and suggest that pharmacological inhibition of PARP can be exploited to target the stem cell component of the TGCTs (namely ECs) and to enhance the sensitivity of cisplatin-resistant TGCTs to standard treatments.

DICER1 RNase IIIb domain mutations are infrequent in testicular germ cell tumours.

BACKGROUND: Testicular Germ Cell Tumours (TGCT) are the most frequently occurring malignancy in males from 15-45 years of age. They are derived from germ cells unable to undergo physiological maturation, although the genetic basis for this is poorly understood. A recent report showed that mutations in the RNase IIIb domain of DICER1, a micro-RNA (miRNA) processing enzyme, are common in non-epithelial ovarian cancers. DICER1 mutations were found in 60% of Sertoli-Leydig cell tumours, clustering in four codons encoding metal-binding sites. Additional analysis of 14 TGCT DNA samples identified one case that also contained a mutation at one of these sites. FINDINGS: A number of previous studies have shown that DICER1 mutations are found in <1% of most cancers. To provide a more accurate estimate of the frequency of such mutations in TGCTs, we have analysed 96 TGCT samples using high resolution melting curve analysis for sequence variants in these four codons. Although we did not detect any mutations in any of these sites, we did identify a novel mutation (c.1725 R>Q) within the RNase IIIb domain in one TGCT sample, which was predicted to disturb DICER1 function. CONCLUSION: Overall our findings suggest a mutation frequency in TGCTs of ~1%. We conclude therefore that hot-spot mutations, frequently seen in Sertoli-Leydig cell tumours, are not common in TGCTs.

Transgenerational epigenetic effects of the Apobec1 cytidine deaminase deficiency on testicular germ cell tumor susceptibility and embryonic viability.

Environmental agents and genetic variants can induce heritable epigenetic changes that affect phenotypic variation and disease risk in many species. These transgenerational effects challenge conventional understanding about the modes and mechanisms of inheritance, but their molecular basis is poorly understood. The Deadend1 (Dnd1) gene enhances susceptibility to testicular germ cell tumors (TGCTs) in mice, in part by interacting epigenetically with other TGCT modifier genes in previous generations. Sequence homology to A1cf, the RNA-binding subunit of the ApoB editing complex, raises the possibility that the function of Dnd1 is related to Apobec1 activity as a cytidine deaminase. We conducted a series of experiments with a genetically engineered deficiency of Apobec1 on the TGCT-susceptible 129/Sv inbred background to determine whether dosage of Apobec1 modifies susceptibility, either alone or in combination with Dnd1, and either in a conventional or a transgenerational manner. In the paternal germ-lineage, Apobec1 deficiency significantly increased susceptibility among heterozygous but not wild-type male offspring, without subsequent transgenerational effects, showing that increased TGCT risk resulting from partial loss of Apobec1 function is inherited in a conventional manner. By contrast, partial deficiency in the maternal germ-lineage led to suppression of TGCTs in both partially and fully deficient males and significantly reduced TGCT risk in a transgenerational manner among wild-type offspring. These heritable epigenetic changes persisted for multiple generations and were fully reversed after consecutive crosses through the alternative germ-lineage. These results suggest that Apobec1 plays a central role in controlling TGCT susceptibility in both a conventional and a transgenerational manner.

Imbalance of MMP-2 and MMP-9 expression versus TIMP-1 and TIMP-2 reflects increased invasiveness of human testicular germ cell tumours.

The histological classification of testicular germ cell tumours (TGCTs) to seminoma or non-seminomatous germ cell tumours is at present the main criterion for the clinical outcome and selection of the treatment strategy. In view of the need to identify novel prognostic biomarkers for TGCTs, we investigated the expression of the matrix metalloproteinases MMP-2 and MMP-9 in testicular tumour tissues and cell lines of both seminoma and non-seminoma origin. Immunohistochemistry and zymography analysis of tumoural tissues showed significantly higher levels of MMP-2 and MMP-9 compared with normal testis with the active forms detected only in the tumour tissues. Three cell lines representative of the different tumour types, JKT-1 seminoma, NCCIT teratocarcinoma and NTERA2/D1 embryonal carcinoma were also evaluated for their expression of these MMPs using qPCR and zymography and for their invasive properties. The more invasive non-seminomatous teratocarcinoma and embryonal cells expressed considerably more MMP-2 and MMP-9 compared with seminoma cells exhibiting lower invasiveness. Furthermore, an inverse relation was observed between invasiveness and the expression of endogenous inhibitors TIMP-1 and TIMP-2. The MMP inhibitor Marimastat inhibited invasion in all cell lines, the highest inhibition was observed in the more invasive NTERA2/D1 and NCCIT cells, which presented the highest ratio of MMP-2 and MMP-9 vs. TIMP-1 and TIMP-2. These results highlight the importance of MMP-2 and MMP-9 in the invasiveness of testicular tumours and suggest that their levels, vs. those of TIMP-1 and TIMP-2, may represent potential biomarkers for testicular malignancy.