Evolution of Testicular Germ Cell Tumors in the Molecular Era With Histogenetic Implications.

The current WHO classification of testicular germ cell tumors is based on the pathogenesis of the tumors driven by different genomic events. The germ cell neoplasia in situ is the precursor lesion for all malignant germ cell tumors. The current understanding of pathogenesis is that the developmental and environmental factors with the erasure of parental genomic imprinting lead to the development of abnormal gonocytes that settle in the "spermatogonial Niche" in seminiferous tubules. The abnormal primordial germ cells in the seminiferous tubules give rise to pre-GCNIS cells under the influence of TPSY and OCT4 genes. The whole genome duplication events give rise to germ cell neoplasia in situ, which further acquires alterations in 12p along with NRAS and KRAS mutations to produce seminoma. A subset of seminomas acquires KIT mutation and does not differentiate further. The remaining KIT-stable seminomas differentiate to nonseminomatous GCTs after obtaining recurrent chromosomal losses, epigenetic modification, and posttranscriptional regulation by multiple genes. Nonseminomatous germ cell tumors also develop directly from differentiated germ cell neoplasia in situ. TP53 pathway with downstream drivers may give rise to somatic-type malignancies of GCT. The GCTs are remarkably sensitive to cisplatin-based combination chemotherapy; however, resistance to cisplatin develops in up to 8% of tumors and appears to be driven by TP53/MDM2 gene mutations. Serum and Plasma miRNAs show promise in diagnosing, managing, and following up on these tumors. The mechanisms underlying the development of most tumors have been elucidated; however, additional studies are required to pinpoint the events directing specific characteristics. Advances in identifying specific molecular markers have been seen recently and may be adopted as gold standards in the future.

Predictive tissue markers in testicular germ cell tumors: Immunohistochemical expression of MLH1 and REV-7 proteins.

PURPOSE: Testicular Germ Cell Tumors (TGCTs) are the most frequent solid malignancies in young adult men. Regardless of differences in their cell of origin, all TGCTs are considered highly curable malignancies. However, approximately 3-5% of all TGCTs do not respond to platinum-based chemotherapies. The purpose of our paper is to investigate whether immunohistochemical expression of MLH1 and REV-7 can be used as predictive tissue markers for TGCTs. MATERIAL AND METHODS: The main demographic and clinicopathological characteristics of 64 male patients with TGCTs who underwent orchiectomy from 2007 to 2022 were retrospectively obtained from two large Oncology Clinics in Greece. Both patients with chemosensitive and chemoresistant disease were included. Immunohistochemical staining for MLH1 and REV-7 proteins was applied in specimens of these patients. RESULTS: 31 seminomas and 33 non-seminomas were included. 48 patients had chemosensitive disease, while 16 had chemoresistant disease. 53 specimens showed preserved MLH1 expression, while 11 specimens had lost MLH1 expression. Expression of MLH1 was only significantly associated with patients' age. 16 specimens showed positive REV-7 expression, while 48 specimens were REV-7 negative. Interestingly, 50% of patients with chemoresistant disease and 16,7% of patients with chemosensitive disease were REV-7 positive. This difference was statistically significant. Moreover, REV-7 positivity was significantly associated with chemoresistance, various clinicopathological parameters and patients' prognosis and survival. CONCLUSION: Loss of MLH1 expression was only found to be significantly associated with lower patients' age. Positive immunohistochemical REV-7 expression was significantly associated with various clinicopathological parameters, while it was also associated with significantly lower survival and greater hazard. REV-7 positive percentages were significantly higher in patients with chemoresistant disease. Our findings imply that immunohistochemical staining for REV-7 could potentially be used as a predictive tissue marker for TGCT tumors. Moreover, targeting of REV-7 protein, could represent a potential therapeutic strategy for chemoresistant TGCT cases. The implementation of well-designed studies on a larger scale is of utmost importance, in order to draw safer conclusions. Additional studies are needed so as to draw safer conclusions.

Single-cell multi-omics analysis of human testicular germ cell tumor reveals its molecular features and microenvironment.

Seminoma is the most common malignant solid tumor in 14 to 44 year-old men. However, its molecular features and tumor microenvironment (TME) is largely unexplored. Here, we perform a series of studies via genomics profiling (single cell multi-omics and spatial transcriptomics) and functional examination using seminoma samples and a seminoma cell line. We identify key gene expression programs share between seminoma and primordial germ cells, and further characterize the functions of TFAP2C in promoting tumor invasion and migration. We also identify 15 immune cell subtypes in TME, and find that subtypes with exhaustion features were located closer to the tumor region through combined spatial transcriptome analysis. Furthermore, we identify key pathways and genes that may facilitate seminoma disseminating beyond the seminiferous tubules. These findings advance our knowledge of seminoma tumorigenesis and produce a multi-omics atlas of in situ human seminoma microenvironment, which could help discover potential therapy targets for seminoma.

MicroRNA as epigenetic regulators of canine cryptorchidism.

Cryptorchidism, the failed descent of one or both testes into the scrotum, is a common developmental disorder in male dogs. Cryptorchidism may affect canine fertility, reducing the quality of the semen, and may promote spermatic cord torsion and onset of neoplasia. MicroRNAs (miRNAs) are epigenetic regulators of gene expression and their dysregulation is associated with disorders of spermatogenesis and testis neoplasia. The present study aimed at investigating the expression of miRNAs in formalin-fixed, paraffin-embedded (FFPE) canine retained testes and testes affected by seminoma, and at integrating miRNAs to their target genes. Forty testicular FFPE specimens from 30 dogs were included - 10 scrotal and 10 contralateral retained from 10 unilateral cryptorchid dogs; 10 tumoral testes affected by seminoma from non-cryptorchid dogs; 10 scrotal normal testes from non-cryptorchid dogs included as the control. The expression level of three miRNAs, namely miR-302c-3p, miR-302a-3p, and miR-371-3p, associated with testicular disorders, were quantified using RT-qPCR. The comparative analysis demonstrated that the level of miR-302a-3p and miR-371a-3p were quantifiable exclusively in control testes. The expression level of miR-302c-3p was higher in the control than in the other groups; its expression decreased in retained testes compared to scrotal testes and testes with seminoma. Gene Ontology analysis pointed out that these miRNAs may be involved in the modulation of estrogen and thyroid hormone signaling pathways. In conclusion, this study demonstrated that miRNAs are dysregulated in canine cryptorchid and seminoma-affected testes compared to control tissues, confirming the pivotal role of miRNAs in cryptorchidism.

Activin and BMP Signalling in Human Testicular Cancer Cell Lines, and a Role for the Nucleocytoplasmic Transport Protein Importin-5 in Their Crosstalk.

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men. Originating from foetal testicular germ cells that fail to differentiate correctly, TGCTs appear after puberty as germ cell neoplasia in situ cells that transform through unknown mechanisms into distinct seminoma and non-seminoma tumour types. A balance between activin and BMP signalling may influence TGCT emergence and progression, and we investigated this using human cell line models of seminoma (TCam-2) and non-seminoma (NT2/D1). Activin A- and BMP4-regulated transcripts measured at 6 h post-treatment by RNA-sequencing revealed fewer altered transcripts in TCam-2 cells but a greater responsiveness to activin A, while BMP4 altered more transcripts in NT2/D1 cells. Activin significantly elevated transcripts linked to pluripotency, cancer, TGF-ß, Notch, p53, and Hippo signalling in both lines, whereas BMP4 altered TGF-ß, pluripotency, Hippo and Wnt signalling components. Dose-dependent antagonism of BMP4 signalling by activin A in TCam-2 cells demonstrated signalling crosstalk between these two TGF-ß superfamily arms. Levels of the nuclear transport protein, IPO5, implicated in BMP4 and WNT signalling, are highly regulated in the foetal mouse germline. IPO5 knockdown in TCam-2 cells using siRNA blunted BMP4-induced transcript changes, indicating that IPO5 levels could determine TGF-ß signalling pathway outcomes in TGCTs.

Extrinsic apoptosis and senescence involved in growth kinetics of seminoma to cisplatin.

Seminoma is the most common type of testicular germ cell tumour and is highly sensitive to cisplatin therapy, which has not been fully elucidated. In this study, we comprehensively monitored dynamic changes of TCam-2 cells after cisplatin treatment. At an early stage, we found that both low and high concentrations of cisplatin induced the S-phase arrest in TCam-2 cells. By contrast, the G0G1 arrest was caused by cisplatin in teratoma NTERA-2 cells. Afterwards, high concentrations of cisplatin promoted the extrinsic apoptosis and high expressions of related genes (Fas/FasL-caspase-8/-3) in TCam-2 cells. However, when decreasing the cisplatin, the apoptotic cells were significantly reduced, and accompanied by cells showing senescence-like morphology, positive SA-ß-gal staining and up-regulation of senescence-related genes (p53, p21 and p16). Furthermore, the cell cycle analysis revealed that most of the senescent TCam-2 cells were irreversibly arrested in the G2M phase. G2M arrest was also observed in NTERA-2 cells treated with a low concentration of cisplatin, while no senescence-related phenotype was discovered. In addition, we detected the γ-H2AX, a DNA damage marker protein, and reactive oxygen species (ROS) and found both of which were elevated with the increase of cisplatin in TCam-2 cells. In conclusion, the extrinsic apoptosis and senescence are involved in the growth kinetics of TCam-2 cells to cisplatin, which provide some implications for studies on cisplatin and seminoma.

PI3K/AKT/mTOR pathway mediated-cell cycle dysregulation contribute to malignant proliferation of mouse spermatogonia induced by microcystin-leucine arginine.

Environmental cyanotoxin exposure may be a trigger of testicular cancer. Activation of PI3K/AKT/mTOR signaling pathway is the critical molecular event in testicular carcinogenesis. As a widespread cyanotoxin, microcystin-leucine arginine (MC-LR) is known to induce cell malignant transformation and tumorigenesis. However, the effects of MC-LR on the regulatory mechanism of PI3K/AKT/mTOR pathway in seminoma, the most common testicular tumor, are unknown. In this study, mouse spermatogonia cell line (GC-1) and nude mice were used to investigate the effects and mechanisms of MC-LR on the malignant transformation of spermatogonia by nude mouse tumorigenesis assay, cell migration invasion assay, western blot, and cell cycle assay, and so forth. The results showed that, after continuous exposure to environmentally relevant concentrations of MC-LR (20 nM) for 35 generations, the proliferation, migration, and invasion abilities of GC-1 cells were increased by 120%, 340%, and 370%, respectively. In nude mice, MC-LR-treated GC-1 cells formed tumors with significantly greater volume (0.998 ± 0.768 cm3 ) and weight (0.637 ± 0.406 g) than the control group (0.067 ± 0.039 cm3 ; 0.094 ± 0.087 g) (P < .05). Furthermore, PI3K inhibitor Wortmannin inhibited the PI3K/AKT/mTOR pathway and its downstream proteins (c-MYC, CDK4, CCND1, and MMP14) activated by MC-LR. Blocking PI3K alleviated MC-LR-induced cell cycle disorder and malignant proliferation, migration and invasive of GC-1 cells. Altogether, our findings suggest that MC-LR can induce malignant transformation of mouse spermatogonia, and the PI3K/AKT/mTOR pathway-mediated cell cycle dysregulation may be an important target for malignant proliferation. This study provides clues to further reveal the etiology and pathogenesis of seminoma.

SOX2 and PRAME in the "reprogramming" of seminoma cells.

BACKGROUND: In recent years, several studies investigated the complex process called "reprogramming" of seminoma (S) cells. The accepted pathogenetic model is a complex network including SOX2, SOX17, OCT3/4 and PRAME, which modulates the epigenetic transcription of numerous downstream genes and drives a divergent gene expression profile resulting in the transition from pure S (P-S) to S component (S-C) of mixed germ cell tumors of the testis (M-GCTT), and finally to embryonal carcinoma (EC). Herein, we tested a large cohort of GCTT with SOX2 and PRAME to evaluate their expression in the evolutionary steps of GCTT and verify if the modulation in the expression of these two molecules could be relevant for the fate of GCTT. METHODS: We tested 43, 19 and 17 consecutive and retrospectively enrolled cases of GCTT, germ cell neoplasia in situ (GCNIS) and uninvolved background testes (UBT), respectively. SOX2 and PRAME expressions have been evaluated with H-score and compared by adopting the appropriate statistic tests (Student's t-test and Mann-Whitney U test). RESULTS: We found that SOX2 was more expressed by nonseminomatous-GCTT (NS-GCTT) (p < 0.001) and EC (p < 0.001) rather than S; by contrast, PRAME showed an opposite expression profile being expressed by S but not by NS-GCTT (p < 0.001) and EC (p < 0.001). S-C showed different expressions of SOX2 and PRAME compared to both P-S (p = 0.002 and <0.001, respectively) and EC (p < 0.001 and 0.042, respectively), with intermediate values between these latter two categories. GCNIS and UBT showed no expression of SOX2 (scattered positive Leydig cells) but high H-score levels of PRAME. CONCLUSIONS: SOX2 and PRAME are differentially expressed and specularly modulated during the "reprogramming" of S cells [P-S (high levels of PRAME, no expression/low levels of SOX2) → S-C (intermediate levels of PRAME, intermediate levels of SOX2) → EC (no expression/low levels of PRAME, high levels of SOX2)], therefore supporting a complex pathogenetic model where the interactions between these two molecules are crucial in determining the fate of GCTT.

Association Study between Polymorphisms in DNA Methylation-Related Genes and Testicular Germ Cell Tumor Risk.

BACKGROUND: Testicular germ cell tumors (TGCT), histologically classified as seminomas and nonseminomas, are believed to arise from primordial gonocytes, with the maturation process blocked when they are subjected to DNA methylation reprogramming. SNPs in DNA methylation machinery and folate-dependent one-carbon metabolism genes have been postulated to influence the proper establishment of DNA methylation. METHODS: In this pathway-focused investigation, we evaluated the association between 273 selected tag SNPs from 28 DNA methylation-related genes and TGCT risk. We carried out association analysis at individual SNP and gene-based level using summary statistics from the Genome Wide Association Study meta-analysis recently conducted by the international Testicular Cancer Consortium on 10,156 TGCT cases and 179,683 controls. RESULTS: In individual SNP analyses, seven SNPs, four mapping within MTHFR, were associated with TGCT risk after correction for multiple testing (q ≤ 0.05). Queries of public databases showed that three of these SNPs were associated with MTHFR changes in enzymatic activity (rs1801133) or expression level in testis tissue (rs12121543, rs1476413). Gene-based analyses revealed MTHFR (q = 8.4 × 10-4), methyl-CpG-binding protein 2 (MECP2; q = 2 × 10-3), and ZBTB4 (q = 0.03) as the top TGCT-associated genes. Stratifying by tumor histology, four MTHFR SNPs were associated with seminoma. In gene-based analysis MTHFR was associated with risk of seminoma (q = 2.8 × 10-4), but not with nonseminomatous tumors (q = 0.22). CONCLUSIONS: Genetic variants within MTHFR, potentially having an impact on the DNA methylation pattern, are associated with TGCT risk. IMPACT: This finding suggests that TGCT pathogenesis could be associated with the folate cycle status, and this relation could be partly due to hereditary factors.

CDKN2AIP-induced cell senescence and apoptosis of testicular seminoma are associated with CARM1 and eIF4ß.

Testicular seminoma is a relatively rare tumor which is mostly detected in male population aged from 15 to 35 years old. Although several molecular biomarkers have been identified to be associated with testicular seminoma pathogenesis, the exact mechanism for testicular seminoma progression remains largely unknown. CDKN2A interacting protein (CDKN2AIP) has previously been identified as a tumor suppressor in multiple malignant diseases. In this study, we aimed to further explore its role in testicular seminoma as well as the underlying molecular mechanisms. Retrospective testicular seminoma clinical samples, normal tissues, NTERA-2 cell line, and mouse xenograft models were used in this study. RT-qPCR, western blot analysis, immunofluorescence microscopy, Co-IP and IP-MS experiments were performed to detect the expression of CDKN2AIP and its interaction with CARM1 and eIF4ß. SA-ß-gal staining assay and H3K9me3 activity experiments were used to subsequently evaluate the cell senescence and apoptosis. Mouse xenograft animal model was used for in vivo study. The results showed that CDKN2AIP is highly expressed in normal testis samples, and is significantly suppressed in testicular seminoma clinical samples and cell line model. Up-regulation of CDKN2AIP is significantly associated with the inhibition of testicular seminoma tumor growth and the increase of cell senescence and apoptosis. CDKN2AIP exhibits anti-tumor activity by interacting with CARM1 and eIF4ß. CDKN2AIP induces testicular seminoma cell senescence by suppressing CARM1 expression and eIF4ß phosphorylation. The CDKN2AIP-CARM1 and CDKN2AIP-eIF4ß interactions, which induce tumor cell senescence and apoptosis, may be the potential druggable molecular pathways in testicular seminoma tumor pathogenesis and progression.

Differential expression of preferentially expressed antigen in melanoma (PRAME) in testicular germ cell tumors - A comparative study with SOX17.

The accurate identification of different components in testicular germ cell tumors (GCT) is essential for tailoring treatment and informing the clinical prognosis. PRAME (preferentially expressed antigen in melanoma), a member in the family of cancer testis antigens, plays critical roles in regulating pluripotency and suppressing somatic/germ cell differentiation in seminomas (SEM). To investigate the potential diagnostic value of PRAME in testicular GCT, here we comparatively examined the expression patterns of PRAME and SOX17 by immunohistochemistry in both pure and mixed GCT. Tissue microarrays constructed from 66 pure or mixed GCT were examined, including 25 seminomas (13 pure and 12 mixed), 35 embryonal carcinomas (EC; 7 pure and 28 mixed), 23 teratomas (TER; 10 pure and 13 mixed), 15 yolk sac tumors (YST; 1 pure and 14 mixed), and 5 choriocarcinomas (CC; 1 pure and 4 mixed), with 11 germ cell neoplasia in situ (GCNIS) and 6 normal testicular tissue as controls. The expression levels of PRAME or SOX17 were evaluated by a scoring system counting for intensity and extent of staining. PRAME nuclear expression was present in 92% (23/25) of SEM, including all 13 pure SEM, and 10 out of 12 seminomatous component of mixed GCT. In contrast, all EC and TER were completely negative for PRAME, and focal expression was demonstrated in 33.3% of YST and 20% of CC. As for SOX17, 96% of SEM and 73% of YST stained positively, whereas EC and CC were negative. Focal nuclear positivity was identified in the epithelial cell component of 17.4% (4/23) of TER. We found the sensitivity of PRAME to detect SEM to be comparable to SOX17, although SOX17 staining is more diffuse and stronger in the majority of cases. The specificity of PRAME for SEM appeared to be superior to that of SOX17 (92% versus 81%). In conclusion, PRAME is preferentially expressed in SEM or within the seminomatous component of mixed GCT with only focal variable expression in YST and CC, but shows no expression in EC and TER. These findings suggest that PRAME can be explored as a diagnostic marker for SEM.

RANKL regulates testicular cancer growth and Denosumab treatment has suppressive effects on GCNIS and advanced seminoma.

BACKGROUND: Testicular germ cell tumours (TGCTs) have a high sensitivity to chemotherapy and a high cure rate, although with serious adverse effects. In the search for tumour suppressive drugs, the RANKL inhibitor Denosumab, used to treat osteoporosis, came up as a candidate since RANKL signalling was recently identified in the testis. METHODS: Expression of RANKL, RANK and OPG, and the effects of RANKL inhibition were investigated in human TGCTs, TGCT-derived cell-lines, and TGCT-xenograft models. Serum RANKL was measured in TGCT-patients. RESULTS: RANKL, RANK, and OPG were expressed in germ cell neoplasia in situ (GCNIS), TGCTs, and TGCT-derived cell lines. RANKL-inhibition reduced proliferation of seminoma-derived TCam-2 cells, but had no effect on embryonal carcinoma-derived NTera2 cells. Pretreatment with Denosumab did not augment the effect of cisplatin in vitro. However, inhibition of RANKL in vivo reduced tumour growth exclusively in the TCam-2-xenograft model and Denosumab-treatment decreased proliferation in human GCNIS cultures. In TGCT-patients serum RANKL had no prognostic value. CONCLUSIONS: This study shows that the RANKL signalling system is expressed in GCNIS and seminoma where RANKL inhibition suppresses tumour growth in vitro and in vivo. Future studies are needed to determine whether RANKL is important for the malignant transformation or transition from GCNIS to invasive tumours.

A Coculture Model Mimicking the Tumor Microenvironment Unveils Mutual Interactions between Immune Cell Subtypes and the Human Seminoma Cell Line TCam-2.

Testicular germ cell cancer (TGCC) is the most common type of cancer in young men. Seminomas account for around half of them and are characterized by a pronounced infiltration of immune cells. So far, the impact of the tumor microenvironment (TME) on disease progression, especially the interaction of individual immune cell subtypes with the tumor cells, remains unclear. To address this question, we used an in vitro TME model involving the seminoma-derived cell line Tcam-2 and immune cell subsets purified from human peripheral blood. T cells and monocytes were strongly activated when individually cocultured with Tcam-2 cells as revealed by increased expression of activation markers and pro-inflammatory cytokines both on the mRNA and protein level. Importantly, the interaction between tumor and immune cells was mutual. Gene expression of pluripotency markers as well as markers of proliferation and cell cycle activity were upregulated in Tcam-2 cells in cocultures with T cells, whereas gene expression of SOX17, a marker for seminomas, was unaltered. Interestingly, the impact of monocytes on gene expression of Tcam-2 cells was less pronounced, indicating that the effects of individual immune cell subsets on tumor cells in the TME are highly specific. Collectively, our data indicate that seminoma cells induce immune cell activation and thereby generate a strong pro-inflammatory milieu, whereas T cells conversely increase the proliferation, metastatic potential, and stemness of tumor cells. Although the employed model does not fully mimic the physiological situation found in TGCC in vivo, it provides new insights potentially explaining the connection between inflammatory infiltrates in seminomas and their tendency to burn out and metastasize.

Transcriptome analysis reveals upregulation of immune response pathways at the invasive tumour front of metastatic seminoma germ cell tumours.

BACKGROUND: Testicular germ cell tumours (TGCTs) have a high metastasis rate. However, the mechanisms related to their invasion, progression and metastasis are unclear. Therefore, we investigated gene expression changes that might be linked to metastasis in seminomatous testicular germ cell tumour (STGCT) patients. METHODS: Defined areas [invasive tumour front (TF) and tumour centre (TC)] of non-metastatic (with surveillance and recurrence-free follow-up >2 years) and metastatic STGCTs were collected separately using laser capture microdissection. The expression of 760 genes related to tumour progression and metastasis was analysed using nCounter technology and validated with quantitative real-time PCR and enzyme-linked immunosorbent assay. RESULTS: Distinct gene expression patterns were observed in metastatic and non-metastatic seminomas with respect to both the TF and TC. Comprehensive pathway analysis showed enrichment of genes related to tumour functions such as inflammation, angiogenesis and metabolism at the TF compared to the TC. Remarkably, prominent inflammatory and cancer-related pathways, such as interleukin-6 (IL-6) signalling, integrin signalling and nuclear factor-κB signalling, were significantly upregulated in the TF of metastatic vs non-metastatic tumours. CONCLUSIONS: IL-6 signalling was the most significantly upregulated pathway in metastatic vs non-metastatic tumours and therefore could constitute a therapeutic target for future personalised therapy. In addition, this is the first study showing intra- and inter-tumour heterogeneity in STGCT.

Overexpression of melanoma-associated antigen A2 has a clinical significance in embryonal carcinoma and is associated with tumor progression.

INTRODUCTION: Melanoma-associated antigen A2 (MAGE-A2) is a member of the cancer-testis antigen family differentially overexpressed in a variety of malignancies and is associated with tumor development. However, clinical significance and prognostic value of MAGE-A2 in different histological subtypes of testicular germ cell tumors (TGCTs) have not been explored. MATERIALS AND METHODS: Here, we aimed to investigate the clinical significance and prognostic impact of MAGE-A2 expression in TGCTs compared to benign tumors as well as adjacent normal tissues and then between seminomas and non-seminomas groups using immunohistochemistry on tissue microarrays. RESULTS: The results indicated a statistically significant difference between overexpression of MAGE-A2 and histological subtypes of TGCTs. A statistically significant association was found between a high level of nuclear expression of MAGE-A2 protein and advanced pT stage (P = 0.022), vascular invasion (P = 0.037), as well as involvement of rete testis (P = 0.022) in embryonal carcinomas. Increased nuclear expression of MAGE-A2 was observed to be associated with more aggressive behaviors and tumor progression rather than cytoplasmic expression in these cases. Further, high level nuclear expression of MAGE-A2 had shorter disease-specific survival (DSS) or progression-free survival (PFS) compared to patients with moderate and low expression of MAGE-A2, however, without a statistically significant association. CONCLUSION: Our results confirm that increased nuclear expression of MAGE-A2 has a clinical significance in embryonal carcinomas and is associated with progression of disease. Moreover, MAGE-A2 may act as a potential predictive biomarker for the prognosis in embryonal carcinomas if follow-up period becomes longer. Further investigations for the biological function of MAGE-A2 are required in future studies.

Immunohistochemical Expression of Preferentially Expressed Antigen in Melanoma (PRAME) in the Uninvolved Background Testis, Germ Cell Neoplasia In Situ, and Germ Cell Tumors of the Testis.

OBJECTIVES: Preferentially expressed antigen in melanoma (PRAME) has a key role in regulating pluripotency of primordial germ cells and in the development of germ cell tumors of the testis (GCTT). However, its immunohistochemical expression in normal testes and its neoplastic counterpart remain largely unknown. METHODS: We retrospectively investigated the expression of PRAME in 26 cases of GCTT, 21 cases of germ cell neoplasia in situ (GCNIS), and 17 cases of uninvolved background testes. RESULTS: We found that PRAME was expressed more strongly by seminomatous rather than nonseminomatous GCTT (P = .000) and by pure seminoma rather than the seminoma component of seminomatous/nonseminomatous GCTT (P = .025). In addition, GCNIS and uninvolved background testes displayed high levels of PRAME expression. CONCLUSIONS: PRAME is an additional marker for the differential diagnosis of GCTT and could play a key role in the transition from seminomatous to nonseminomatous GCTT.

Comprehensive characteristics of pathological subtypes in testicular germ cell tumor: Gene expression, mutation and alternative splicing.

Background: Testicular germ cell tumor (TGCT) is the most common tumor in young men, but molecular signatures, especially the alternative splicing (AS) between its subtypes have not yet been explored. Methods: To investigate the differences between TGCT subtypes, we comprehensively analyzed the data of gene expression, alternative splicing (AS), and somatic mutation in TGCT patients from the TCGA database. The gene ontology (GO) enrichment analyses were used to explore the function of differentially expressed genes and spliced genes respectively, and Spearman correlation analysis was performed to explore the correlation between differential genes and AS events. In addition, the possible patterns in which AS regulates gene expression were elaborated by the ensemble database transcript atlas. And, we identified important transcription factors that regulate gene expression and AS and functionally validated them in TGCT cell lines. Results: We found significant differences between expression and AS in embryonal carcinoma and seminoma, while mixed cell tumors were in between. GO enrichment analyses revealed that both differentially expressed and spliced genes were enriched in transcriptional regulatory pathways, and obvious correlation between expression and AS events was determined. By analyzing the transcript map and the sites where splicing occurs, we have demonstrated that AS regulates gene expression in a variety of ways. We further identified two pivot AS-related molecules (SOX2 and HDAC9) involved in AS regulation, which were validated in embryonal carcinoma and seminoma cell lines. Differences in somatic mutations between subtypes are also of concern, with our results suggesting that mutations in some genes (B3GNT8, CAPN7, FAT4, GRK1, TACC2, and TRAM1L1) occur only in embryonal carcinoma, while mutations in KIT, KARS, and NRAS are observed only in seminoma. Conclusions: In conclusion, our analysis revealed the differences in gene expression, AS and somatic mutation among TGCT subtypes, providing a molecular basis for clinical diagnosis and precise therapy of TGCT patients.

Preliminary Investigation on the Involvement of Cytoskeleton-Related Proteins, DAAM1 and PREP, in Human Testicular Disorders.

Herein, for the first time, the potential relationships between the cytoskeleton-associated proteins DAAM1 and PREP with different testicular disorders, such as classic seminoma (CS), Leydig cell tumor (LCT), and Sertoli cell-only syndrome (SOS), were evaluated. Six CS, two LCT, and two SOS tissue samples were obtained during inguinal exploration in patients with a suspect testis tumor based on clinical examination and ultrasonography. DAAM1 and PREP protein levels and immunofluorescent localization were analyzed. An increased DAAM1 protein level in CS and SOS as compared to non-pathological (NP) tissue was observed, while LCT showed no significant differences. Conversely, PREP protein level increased in LCT, while it decreased in CS and SOS compared to NP tissue. These results were strongly supported by the immunofluorescence staining, revealing an altered localization and signal intensity of DAAM1 and PREP in the analyzed samples, highlighting a perturbed cytoarchitecture. Interestingly, in LCT spermatogonia, a specific DAAM1 nuclear localization was found, probably due to an enhanced testosterone production, as confirmed by the increased protein levels of steroidogenic enzymes. Finally, although further studies are needed to verify the involvement of other formins and microtubule-associated proteins, this report raised the opportunity to indicate DAAM1 and PREP as new potential markers, supporting the cytoskeleton dynamics changes occurring during normal and/or pathological cell differentiation.

On the origin of germ cell neoplasia in situ: Dedifferentiation of human adult Sertoli cells in cross talk with seminoma cells in vitro.

Germ cell neoplasia in situ (GCNIS) is the noninvasive precursor of testicular germ cell tumors type II, the most common cancer in young men, which originates from embryonic germ cells blocked in their maturation. GCNIS is associated with impaired Sertoli cells (SCs) that express fetal keratin 18 (KRT18) and the pluripotency factor SRY-Box transcription factor 2 (SOX2). According to the current theory concerning the origin of GCNIS, these SCs are prepubertal cells arrested in their maturation due to (epi)genetic anomalies and/or environmental antiandrogens. Thus, they are unable to support the development of germ cells, which leads to their maturational block and further progresses into GCNIS. Alternatively, these SCs are hypothesized to be adult cells dedifferentiating secondarily under the influence of GCNIS. To examine whether tumor cells can dedifferentiate SCs, we established a coculture model of adult human SCs (FS1) and a seminoma cell line similar to GCNIS (TCam-2). After 2 wk of coculture, FS1 cells showed progressive expression of KRT18 and SOX2, mimicking the in vivo changes. TCam-2 cells showed SOX2 expression and upregulation of further pluripotency- and reprogramming-associated genes, suggesting a seminoma to embryonal carcinoma transition. Thus, our FS1/TCam-2 coculture model is a valuable tool for investigating interactions between SCs and seminoma cells. Our immunohistochemical and ultrastructural studies of human testicular biopsies with varying degrees of GCNIS compared to biopsies from fetuses, patients with androgen insensitivity syndrome, and patients showing normal spermatogenesis further suggest that GCNIS-associated SCs represent adult cells undergoing progressive dedifferentiation.

Methylation gene KCNC1 is associated with overall survival in patients with seminoma.

The aim of the present study was to explore and verify the potential mechanism of seminoma progression. Data on 132 RNA­seq and 156 methylation sites from stage II/III and I seminoma specimens were downloaded from The Cancer Genome Atlas database. An initial filter of |fold­change| >2 and false discovery rate <0.05 were used to identify differentially expressed genes (DEGs) which were associated with differential methylation site genes; these genes were considered potential candidates for further investigation by survival analysis. Potassium voltage­gated channel subfamily C member 1 (KCNC1) expression was verified in seminoma human tissues and three seminoma cell lines. The invasive, proliferative and apoptotic abilities of the human testicular tumor Ntera­2 and normal human testis Hs1.Tes cell lines were assessed following aberrant KCNC1 expression. KCNC1 was identified as a DEG, in which hypermethylation inhibited its expression and it was associated with poor overall survival in patients with seminoma. The present results demonstrated that KCNC1 is negatively correlated with methylation. Due to the abnormal expression of KCNC1 in seminoma cells, it was suggested that KCNC1 could be used as a diagnostic indicator and therapeutic target for the progression of seminoma.