New analysis of atypical spermatocytic tumours reveals extensive heterogeneity and plasticity of germ cell tumours†.

Testicular germ cell tumours (TGCTs) derived from immature (type I) and pluripotent germ cell neoplasia in situ (GCNIS, type II) are characterised by remarkable phenotypic heterogeneity and plasticity. In contrast, the rare spermatocytic tumour (SpT, type III), derived from mature spermatogonia, is considered a homogenous and benign tumour but may occasionally present as an anaplastic or an aggressive sarcomatoid tumour. While various oncogenic processes had been proposed, the precise mechanism driving malignant progression remained elusive until the molecular characterisation of a series of atypical SpTs described in a recent issue of The Journal of Pathology. The emerging picture suggests the presence of two distinct trajectories for SpTs, involving either RAS/mitogen-activated protein kinase pathway mutations or a ploidy shift with secondary TP53 mutations and/or gain of chromosome 12p, the latter known as pathognomonic for type II GCNIS-derived TGCTs. Here, we discuss the implications of these findings, seen from the perspective of germ cell biology and the unique features of different TGCTs. The evolving phenotype of SpTs, induced by genomic and epigenetic changes, illustrates that the concept of plasticity applies to all germ cell tumours, making them inherently heterogenous and capable of significant transformation during progression. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Single-cell mRNA sequencing of giant panda (Ailuropoda melanoleuca) seminoma reveals the cellular and molecular characteristics of tumour cells.

Testicular tumours are zoonoses that can occur in not only human, but other animals, include giant pandas. A middle-aged male giant panda named Fufu was diagnosed with a testicular tumour and underwent surgery to remove the entire left testis. The testis was mainly composed of three substantive parts: normal tissue on the outside, tumour tissue in the middle, and necrosis in the centre. HE stains revealed that the tumour was a seminoma. Single-cell mRNA sequence was applied to characterise cellular states and molecular circuitries of giant panda testicular seminoma. Only germ cell markers expressed in nearly all tumour cells, and the tumour cells appeared to be the same subtype of seminoma cells. We identified four clusters with unique genes expression. They were early apoptosis cells (EAC), inactive cells (IC), active cells subcluster 1 (AC-1) and active cells subcluster 2 (AC-2). We utilised monocle tools and found that IC cells was in the initiation stage, and EAC was one type of terminal stage, suggesting that tumour cells may undergo apoptosis in the future. AC-2 was another type of terminal stage, representing a group of progressive cells. Our study represents the first report to utilise scRNA-seq to characterise the cellular states and molecular circuitries of a giant panda testicular tumour. This investigation proposes CD117 and CD30 as dependable markers for future pathologic diagnosis. Our findings also suggest that CTSV and other genes with unique expression patterns in active and progressive giant panda seminoma cells may act as early prognostic biomarkers.

Genomic analysis of spermatocytic tumors demonstrates recurrent molecular alterations in cases with malignant clinical behavior.

Spermatocytic tumor (ST) is a rare type of germ cell tumor that occurs exclusively in the postpubertal testis and typically affects elderly men. Most STs are benign, but rare cases exhibit aggressive clinical behavior, often in association with transition to sarcomatoid histology. Limited molecular analyses have been performed on STs; therefore, their genomic and epigenomic features remain incompletely described. Twenty-seven samples from 25 individual patients were analyzed with a combination of DNA sequencing panels, genomic methylation profiling, SNP array, isochromosome (12p) [i(12p)] FISH, and immunohistochemistry. The series included five metastasizing tumors (three with sarcomatoid transformation, one anaplastic, and one conventional) and 20 non-metastasizing tumors (14 anaplastic and six conventional). Anaplastic tumors comprised a monomorphic population of intermediate-sized neoplastic cells, as previously described. Multiomic analyses demonstrated that there were two genomic subgroups of STs: one with diploid genomes and hotspot RAS/RAF variants and the other with global ploidy shift and absence of recurrent mutations. Relative gain of chromosome 9 was a consistent finding in both subgroups. A comparison of metastasizing and non-metastasizing cases demonstrated that aggressive behavior was associated with the acquisition of pathogenic TP53 mutations and/or relative gains of 12p/i(12p). In cases with sarcomatoid transformation, TP53 mutations seem to underlie the transition to sarcomatoid histology. Genomic methylation analysis demonstrated that aggressive cases with gains of 12p cluster closer to pure seminomas than to STs without gains of 12p. In conclusion, STs include two genomic subgroups, characterized by global ploidy shifts without recurrent mutations and diploid genomes with RAS/RAF hotspot mutations, respectively. Biologic progression was associated with relative gains of 12p and TP53 mutations. The findings in STs with relative gains of 12p suggest that they may exhibit biologic characteristics akin to those seen in germ cell neoplasia in situ-related germ cell tumors rather than non-germ cell neoplasia in situ-derived STs. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

SPTBN1 Mediates the Cytoplasmic Constraint of PTTG1, Impairing Its Oncogenic Activity in Human Seminoma.

Seminoma is the most common testicular cancer. Pituitary tumor-transforming gene 1 (PTTG1) is a securin showing oncogenic activity in several tumors. We previously demonstrated that nuclear PTTG1 promotes seminoma tumor invasion through its transcriptional activity on matrix metalloproteinase 2 (MMP-2) and E-cadherin (CDH1). We wondered if specific interactors could affect its subcellular distribution. To this aim, we investigated the PTTG1 interactome in seminoma cell lines showing different PTTG1 nuclear levels correlated with invasive properties. A proteomic approach upon PTTG1 immunoprecipitation uncovered new specific securin interactors. Western blot, confocal microscopy, cytoplasmic/nuclear fractionation, sphere-forming assay, and Atlas database interrogation were performed to validate the proteomic results and to investigate the interplay between PTTG1 and newly uncovered partners. We observed that spectrin beta-chain (SPTBN1) and PTTG1 were cofactors, with SPTBN1 anchoring the securin in the cytoplasm. SPTBN1 downregulation determined PTTG1 nuclear translocation, promoting its invasive capability. Moreover, a PTTG1 deletion mutant lacking SPTBN1 binding was strongly localized in the nucleus. The Atlas database revealed that seminomas that contained higher nuclear PTTG1 levels showed significantly lower SPTBN1 levels in comparison to non-seminomas. In human seminoma specimens, we found a strong PTTG1/SPTBN1 colocalization that decreases in areas with nuclear PTTG1 distribution. Overall, these results suggest that SPTBN1, along with PTTG1, is a potential prognostic factor useful in the clinical management of seminoma.

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.

Targeting oncogenic microRNAs from the miR-371~373 and miR-302/367 clusters in malignant germ cell tumours causes growth inhibition through cell cycle disruption.

BACKGROUND: MiR-371~373 and miR-302/367 cluster over-expression occurs in all malignant germ cell tumours (GCTs), regardless of age (paediatric/adult), site (gonadal/extragonadal), or subtype [seminoma, yolk sac tumour (YST), embryonal carcinoma (EC)]. Six of eight microRNAs from these clusters contain the seed sequence 'AAGUGC', determining mRNA targeting. Here we sought to identify the significance of these observations by targeting these microRNAs functionally. METHODS: We targeted miR-371~373 and/or miR-302/367 clusters in malignant GCT cell lines, using CRISPR-Cas9, gapmer primary miR-302/367 transcript inhibition, and peptide nucleic acid (PNA) or locked nucleic acid (LNA)-DNA inhibition targeting miR-302a-d-3p, and undertook relevant functional assays. RESULTS: MiR-302/367 cluster microRNAs made the largest contribution to AAGUGC seed abundance in malignant GCT cells, regardless of subtype (seminoma/YST/EC). Following the unsuccessful use of CRISPR-Cas9, gapmer, and PNA systems, LNA-DNA-based targeting resulted in growth inhibition in seminoma and YST cells. This was associated with the de-repression of multiple mRNAs targeted by AAGUGC seed-containing microRNAs, with pathway analysis confirming predominant disruption of Rho-GTPase signalling, vesicle organisation/transport, and cell cycle regulation, findings corroborated in clinical samples. Further LNA-DNA inhibitor studies confirmed direct cell cycle effects, with an increase of cells in G0/G1-phase and a decrease in S-phase. CONCLUSION: Targeting of specific miR-371~373 and miR-302/367 microRNAs in malignant GCTs demonstrated their functional significance, with growth inhibition mediated through cell cycle disruption.

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.

Testicular neoplasms: the interrelationships of serum levels of microRNA-371a-3p (M371) and classical tumor markers with histology, clinical staging, and age-a statistical analysis.

PURPOSE: In testicular neoplasms, the interrelationship of elevations of the novel serum tumor marker microRNA-371a-3p (M371) and traditional markers with other clinical features is still incompletely understood. The present study evaluated marker expression rates in relation to various other clinical parameters. METHODS: The following data were retrospectively registered from 641 consecutive patients with testicular neoplasms: histology, such as seminoma (n = 365), nonseminoma (n = 179), benign tumor (n = 79), other malignant tumor (n = 18); patients age (years); clinical stage (CS1, CS2a/b, CS2c, CS3); and preoperative elevation of beta HCG, AFP, LDH, M371 (yes/no). Descriptive statistical methods were employed with comparisons of various subgroups to disclose associations of marker expression rates with age, histology and CS, and of age with histology. RESULTS: The histologic subgroups revealed significantly different expression rates of tumor markers. M371 performed best with expression rates of 82.69% and 93.58% in seminoma and in nonseminoma, respectively. In germ cell tumors, all markers had significantly higher expression rates in metastasized stages than in localized disease. All markers except LDH have significantly higher expression rates in younger than in older patients. Nonseminoma is most prevalent in the youngest age category, seminoma predominates in patients > 40 years, other malignancies were restricted to patients > 50 years. CONCLUSION: The study documented significant associations of serum marker expression rates with histology, age and clinical staging, with highest rates in nonseminomas, young age and advanced clinical stages. M371 showed significantly higher expression rates than other markers suggesting its superior clinical usefulness.

Inflammatory and Nested Testicular Sex Cord Tumor: A Novel Neoplasm With Aggressive Clinical Behavior and Frequent EWSR1::ATF1 Gene Fusions.

A subset of malignant testicular sex cord tumors (TSCTs), heretofore interpreted as Sertoli cell tumors, not otherwise specified, exhibits distinctive morphologic features that partially overlap with those of seminoma. In this study, we evaluated the clinicopathologic and molecular characteristics of 13 such tumors. The patients were 20 to 73 years old (median, 36 y), and all with available data presented with testicular masses (median size, 3 cm), with 2 having synchronous retroperitoneal metastases. All 11 patients with available follow-up developed metastases to retroperitoneal lymph nodes, nonretroperitoneal lymph nodes, bone, contralateral testis, and/or lung. Microscopically, the tumors showed solid nests and sheets of epithelioid cells with granular, eosinophilic to clear/vacuolated cytoplasm, admixed in most (12/13) cases with variable proportions of lymphocytes, plasma cells, eosinophils, and neutrophils. Additional features included intracytoplasmic hyaline inclusions and a prominent collagenous, sometimes hyalinized stroma. Mitotic activity was relatively low (median, 1 mitosis/10 HPF), but tumor necrosis was frequent (11/13). Local invasion of adjacent structures and lymphovascular invasion were noted in some tumors (4/9 cases with available data for each feature). All were α-inhibin-positive and lacked nuclear reactivity for ß-catenin. In addition, all tested cases were positive for epithelial membrane antigen (9/9) and steroidogenic factor-1 (8/8), and 8/10 expressed CD30. Two "index" cases were initially analyzed using a DNA sequencing panel, which identified EWSR1::ATF1 fusions in both. Subsequently, EWSR1::ATF1 fusions were demonstrated in 8 of the remaining 11 cases using fluorescence in situ hybridization or DNA sequencing. One of the 3 cases that were negative for EWSR1::ATF1 harbored ATF1 amplification. This study, therefore, shows that a group of malignant TSCTs resembling seminoma is characterized by α-inhibin and steroidogenic factor-1 positivity, no expression of nuclear ß-catenin, frequent CD30 positivity and recurrent EWSR1::ATF1 fusions. We have descriptively termed these neoplasms "inflammatory and nested TSCT." Importantly, inflammatory and nested TSCTs show significant differences in morphology, immunoprofile, molecular biology, and, likely, clinical behavior from Sertoli cell tumors, not otherwise specified and should be classified separately.

Germ cell neoplasms of the testis: Update for 2022.

Germ cell neoplasia in situ (GCNIS) is the precursor of both seminomatous and non-seminomatous germ cell tumors. It consists of distended tubules that may have either intratubular seminoma or intratubular embryonal carcinoma cells. Many invasive non-seminomatous tumors contain a mixture of tumor types, which are reviewed here. Morphology, aided by a panel of immunostains, can determine the presence and percent of embryonal carcinoma, yolk sac tumor, choriocarcinoma, or teratoma in such tumors. Use of immunostains, required for diagnosis in perhaps 25% of testicular neoplasms, is reviewed. Changes of classification in the AJCC (8th edition) in 2016 are discussed, including the partitioning of two tumor types: the central role of chromosome 12p amplification allows both teratoma and yolk sac tumor to be divided into prepubertal types (lacking amplification) and post-pubertal types. Occasionally, sex cord-stromal tumors, hematolymphoid tumors, or epididymal adenomatoid tumors enter the differential diagnosis of germ cell neoplasms.

Evaluation of miR-371a-3p to predict viable germ cell tumor in patients with pure seminoma receiving retroperitoneal lymph node dissection.

INTRODUCTION AND OBJECTIVE: Conventional serum tumor markers (STMs) for testicular germ cell tumors (GCTs) offer limited performance with particularly poor sensitivity in cases of minimal residual disease and pure seminoma. While growing evidence has indicated miR-371a-3p to be a superior biomarker, its utility in detecting pure seminoma at recurrence has not been extensively explored. This study's objective was to explore miR-371a-3p's utility in detecting metastatic pure seminoma at retroperitoneal lymph node dissection (RPLND). METHODS: RNA was isolated from patient serum samples collected pre-RPLND. Fifteen patients were assigned to our 'benign' (n = 6) or 'seminoma' (n = 9) group based on pathological confirmation of viable seminoma. Five of the patients received chemotherapy before RPLND (PC-RPLND), and 10 were chemotherapy naïve. MiR-371a-3p expression was quantified via RT-quantitative polymerase chain reaction. The Cq values were statistically evaluated to obtain performance measurements. RESULTS: Median relative expression of miR-371a-3p was higher in the Seminoma group than the Benign, but this difference was not statistically significant (Rq = 3705 and 241, respectively, p = 0.2844). Of the 10 chemotherapy naïve patients, nine had viable seminoma at RPLND, and seven had elevated miR-371a-3p expression. Among the five postchemotherapy (PC) patients, zero had viable GCT at RPLND, and two had elevated miR-371a-3p expression. The primary RPLND group presented 78% sensitivity and 100% specificity. Specificity in the PC-RPLND group was 60%. An optimal Rq threshold of 28.62 was determined by Youden's J statistic, yielding 78% sensitivity and 67% specificity. Receiver operating characteristic analysis provided an AUC of 0.704 (95% CI: 0.43-0.98, p = 0.1949). Despite modest performance, miR-371a-3p exhibited improved sensitivity and specificity compared with conventional STMs. CONCLUSIONS: MiR-371a-3p outperformed STMs in the primary RPLND settings. However, miR-371a-3p was not a robust predictor of pathology in the PC setting. These results suggest that pure seminoma at RPLND is a clinical context, wherein the miRNA assay may require further refinement.

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.

Exosomal miR-193b-3p Contributes to Cisplatin Sensitivity in Seminoma by Targeting ZBTB7A.

A previous study confirmed that miRNAs play an important role in the chemosensitivity of seminoma. Increasing evidence reveals that exosomes participate in the regulation of cisplatin resistance by carrying miRNAs. In this study, we further explored whether exosomes regulated the chemosensitivity of seminoma TCam-2 cells to cisplatin. Initially, cisplatin-resistant TCam-2 cells were induced. Our results revealed that exosomes from cisplatin-resistant TCam-2 cells (rExos) could affect the viability of TCam-2 cells in the context of cisplatin treatment through regulation of both cell apoptosis and the cell cycle. Meanwhile, the levels of γ-H2AX were negatively modulated by rExos, which indicated that rExos could decrease the DNA damage from cisplatin. Furthermore, miR-193b-3p was enriched in rExos, and exosomal miR-193b-3p enhanced the proliferative ability of TCam-2 cells under cisplatin treatment. Mechanistically, exosomal miR-193b-3p targets ZBTB7A, which further decreases apoptosis and promotes cell cycle progression. Taken together, these findings indicate that exosomal miR-193b-3p regulates the chemosensitivity of TCam-2 cells to cisplatin through ZBTB7A signaling and could be a promising drug target for patients with chemoresistant 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.

Transcriptome and methylome analysis of CNS germ cell tumor finds its cell-of-origin in embryogenesis and reveals shared similarities with testicular counterparts.

BACKGROUND: CNS germ cell tumors (GCTs) predominantly develop in pediatric and young adult patients with variable responses to surgery, radiation, and chemotherapy. This study aimed to examine the complex and largely unknown pathogenesis of CNS GCTs. METHODS: We used a combined transcriptomic and methylomic approach in 84 cases and conducted an integrative analysis of the normal cells undergoing embryogenesis and testicular GCTs. RESULTS: Genome-wide transcriptome analysis in CNS GCTs indicated that germinoma had a transcriptomic profile representative of primitive cells during early embryogenesis with high meiosis/mitosis potentials, while nongerminomatous GCTs (NGGCTs) had differentiated phenotypes oriented toward tissue formation and organogenesis. Co-analysis with the transcriptome of human embryonic cells revealed that germinomas had expression profiles similar to those of primordial germ cells, while the expression profiles of NGGCTs were similar to those of embryonic stem cells. Some germinoma cases were characterized by extensive immune-cell infiltration and high expression of cancer-testis antigens. NGGCTs had significantly higher immune-cell infiltration, characterized by immune-suppression phenotype. CNS and testicular GCTs (TGCTs) had similar mutational profiles; TGCTs showed enhanced copy number alterations. Methylation analysis clustered germinoma/seminoma and nongerminoma/nonseminoma separately. Germinoma and seminoma were co-categorized based on the degree of the tumor microenvironment balance. CONCLUSIONS: These results suggested that the pathophysiology of GCTs was less dependent on their site of origin and more dependent on the state of differentiation as well as on the tumor microenvironment balance. This study revealed distinct biological properties of GCTs, which will hopefully lead to future treatment development.

Whole Genome Sequencing Reveals Independent Clonal Origin of Bilateral Testicular Germ Cell Tumors in 2 Patients with Pure Seminoma.

The pathophysiology of bilateral testicular germ cell tumors (TGCT) is poorly understood. It is unclear if they develop independently, arise from common germline genetic changes, or metastasize from one gonad to the other. We determined the underlying genomic alterations in two cases of bilateral TGCTs with pure seminoma using whole genome sequencing. Large chromosomal aberrations and KIT amplification were identified, but there were no shared single nucleotide variants or structural chromosomal rearrangements in paired TGCTs, suggesting they develop independently. The biological behavior of bilateral TGCTs may be distinct and the staging and prognostic evaluation of each tumor should be performed independently.