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.

Frequent expression of CD45RO memory T cell marker as well as low to high expression of PD-1 and PD-L1 inhibitory molecules in seminoma and dysgerminoma.

BACKGROUND: Seminoma and dysgerminoma are rare testicular and ovarian germ cell tumors characterized by a significant infiltration of immune cells in the tumor microenvironment. According to the failure of conventional treatments in some patients, it is crucial to identify novel prognostic and therapeutic biomarkers for these patients. The objectives of this study were to evaluate the expression of CD45RO and PD-1/PD-L1 and investigate their association with the clinicopathological characteristics of the patients. METHODS: Immunohistochemistry was performed to assess the expression of CD45RO, PD-1, and PD-L1 in tumor-infiltrated lymphocytes (TILs), and tumor cells in 33 seminoma and 31 dysgerminoma patients. The expression levels were evaluated using a semiquantitative approach, weighted histoscore, which considers both the intensity and extent of staining. RESULTS: All seminoma and dysgerminoma patients exhibited CD45RO expression in TILs, with 66.7 % and 90.3 % displaying high levels of expression, respectively. PD-1 expression in TILs was observed at low levels in 81.8 % and 77.4 % and at high levels in 18.2 % and 19.4 % of seminoma and dysgerminoma patients, respectively. Likewise, low expression of PD-L1 in tumor cells was detected in 63.6 % of seminoma and 61.3 % of dysgerminoma patients, while none of the patients exhibited high expression of PD-L1. In seminoma patients, a positive correlation was observed between PD-1 expression in TILs and CD45RO expression and between PD-L1 expression in tumor cells and TILs score. CONCLUSION: The frequent infiltration of CD45RO, along with variable expression of PD-1 and PD-L1 on TILs and tumor cells, could impact the effectiveness of anti-tumor responses and immunotherapy.

Overexpression of SerpinB9 in non-seminomatous germ cell tumors.

Serpinb9 is an inhibitor of granzyme B and is potentially involved in the immune escape of tumor cells. In the present study, bioinformatics analysis using open databases suggested that SerpinB9 is overexpressed in testicular embryonal carcinoma. Immunohistological analysis was performed on 28 cases of testicular germ cell tumors to investigate the relationship between SerpinB9 expression in testicular germ cell tumors and the tumor immune environment. SerpinB9 was significantly upregulated in the non-seminoma group and inversely correlated with the number of tumor-infiltrating CD8-positive cells. In addition, yolk sac tumors were characterized by the loss of human leukocyte antigen-class I expression. These findings suggest that SerpinB9 contributes to the immune escape of testicular germ cell tumors. Targeting therapy for SerpinB9 might therefore be useful in immunotherapy for testicular germ cell tumors resistant to immune checkpoint inhibitors.

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.

Sertoli Cell-Specific Activation of Transforming Growth Factor Beta Receptor 1 Leads to Testicular Granulosa Cell Tumor Formation.

The transforming growth factor ß (TGFß) superfamily, consisting of protein ligands, receptors, and intracellular SMAD transducers, regulates fundamental biological processes and cancer development. Our previous study has shown that sustained activation of TGFß receptor 1 (TGFBR1) driven by anti-Mullerian hormone receptor type 2 (Amhr2)-Cre in the mouse testis induces the formation of testicular granulosa cell tumors (TGCTs). As Amhr2-Cre is expressed in both Sertoli cells and Leydig cells, it remains unclear whether the activation of TGFBR1 in Sertoli cells alone is sufficient to induce TGCT formation. Therefore, the objective of this study was to determine whether Sertoli cell-activation of TGFBR1 drives oncogenesis in the testis. Our hypothesis was that overactivation of TGFBR1 in Sertoli cells would promote their transdifferentiation into granulosa-like cells and the formation of TGCTs. To test this hypothesis, we generated mice harboring constitutive activation of TGFBR1 in Sertoli cells using anti-Mullerian hormone (Amh)-Cre. Disorganized seminiferous tubules and tumor nodules were found in TGFBR1CA; Amh-Cre mice. A histological analysis showed that Sertoli cell-specific activation of TGFBR1 led to the development of neoplasms resembling granulosa cell tumors, which derailed spermatogenesis. Moreover, TGCTs expressed granulosa cell markers including FOXL2, FOXO1, and INHA. Using a dual fluorescence reporter line, the membrane-targeted tdTomato (mT)/membrane-targeted EGFP (mG) mouse, we provided evidence that Sertoli cells transdifferentiated toward a granulosa cell fate during tumorigenesis. Thus, our findings indicate that Sertoli cell-specific activation of TGFBR1 leads to the formation of TGCTs, supporting a key contribution of Sertoli cell reprogramming to the development of this testicular malignancy in our model.

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.

Administration of a glypican-3 peptide increases the infiltration and cytotoxicity of CD8+ T cells against testicular yolk sac tumor, associated with enhancing the intratumoral cGAS/STING signaling.

BACKGROUND: Glypican-3 (GPC3) is highly expressed in testicular yolk sac tumor (TYST). GPC3 has been evaluated as a cancer vaccine for some types of tumors, but little is known on the effects of GPC3 peptide-based therapy on TYST. Here, we evaluated the antitumor effect of GPC3144-152 on TYST and its potential mechanisms. METHODS: GPC3144-152 -specific CD8+ T cells were induced by vaccine immunization and examined by ELISPOT. The CD8+ T cells were purified for testing their cytotoxicity in vitro against TYST cells by CCK-8 and TUNEL assays and in vivo against tumor growth. The influence of GPC3144-152 loading and/or cGAS silencing on the tumor growth, apoptosis and cGAS/STING signaling was tested by immunohistochemistry, immunofluorescence, flow cytometry, and Western blot. RESULTS: Vaccination with GPC3144-152 induced tumor-specific CD8+ T cells that secreted high levels of IFN-γ and granzyme B, and had potent cytotoxicity against TYST in a dose-dependent manner. Adoptive transfer of CD8+ T cells and treatment with GPC3144-152 significantly inhibited the growth of TYST tumors, but less effective for cGAS-silenced TYST tumors in vivo. Treatment with GPC3144-152 enhanced the infiltration of CD8+ T cells into the tumor environment and their cytotoxicity against TYST tumors in vivo by up-regulating granzyme B and IFN-ß expression, but down-regulating GPC3 expression in the tumors. Co-culture of CD8+ T cells with TYST in the presence of exogenous GPC3144-152 enhanced peptide-specific CD8+ T-cell cytotoxicity in vitro, accompanied by enhancing cGAS, γH2AX, TBK1, and IRF3 phosphorylation in TYST cells, but less effective in cGAS-silenced TYST cells. CONCLUSIONS: These data indicated that GPC3 peptide-specific CD8+ T cells had potent antitumor activity against TYST tumor, particularly for combined treatment with the peptide, which was partially dependent on the intratumoral cGAS/STNG signaling. GPC3 peptide vaccine may be valuable for the combination treatment of TYST.

Evaluation of Ex Vivo Adrenocorticotropic Hormone Responsiveness of Human Fetal Testis.

Testicular adrenal rest tumors (TARTs), commonly occurring in males with congenital adrenal hyperplasia, may arise from chronic stimulation of adrenocorticotropic hormone (ACTH)-sensitive cells in the testes. It is not yet established whether the human fetal testis (HFT) is responsive to ACTH. To investigate this, we cultured HFT tissue with and without ACTH for up to 5 days, and quantified adrenal steroid hormones and expression of adrenal steroidogenic enzymes. Fetal testis and adrenal tissue produced high levels of testosterone and cortisol, respectively, indicating viability. In contrast to fetal adrenal tissues, the expression of ACTH receptor MC2R was either absent or expressed at extremely low levels in ex vivo HFT tissue and no clear response to ACTH in gene expression or steroid hormone production was observed. Altogether, this study suggests that the HFT is unresponsive to ACTH, which would indicate that a TART does not arise from fetal testicular cells chronically exposed to ACTH in utero.

X-linked RBBP7 mutation causes maturation arrest and testicular tumors.

Maturation arrest (MA) is a subtype of non-obstructive azoospermia, and male infertility is a known risk factor for testicular tumors. However, the genetic basis for many affected individuals remains unknown. Here, we identified a deleterious hemizygous variant of X-linked retinoblastoma-binding protein 7 (RBBP7) as a potential key cause of MA, which was also found to be associated with the development of Leydig cell tumors. This mutation resulted in premature protein translation termination, affecting the sixth WD40 domain of the RBBP7 and the interaction of the mutated RBBP7 with histone H4. Decreased BRCA1 and increased γH2AX were observed in the proband. In mouse spermatogonial and pachytene spermatocyte-derived cells, deprivation of rbbp7 led to cell cycle arrest and apoptosis. In Drosophila, knockdown of RBBP7/Caf1-55 in germ cells resulted in complete absence of germ cells and reduced testis size, whereas knockdown of RBBP7/Caf1-55 in cyst cells resulted in hyperproliferative testicular cells. Interestingly, male infertility caused by Caf1-55 deficiency was rescued by ectopic expression of wild-type human RBBP7 but not mutant variants, suggesting the importance of RBBP7 in spermatogenesis. Our study provides insights into the mechanisms underlying the co-occurrence of MA and testicular tumors and may pave the way for innovative genetic diagnostics of these 2 diseases.

The Molecular Mechanism and Therapeutic Application of Autophagy for Urological Disease.

Autophagy is a lysosomal degradation process known as autophagic flux, involving the engulfment of damaged proteins and organelles by double-membrane autophagosomes. It comprises microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. Macroautophagy consists of three stages: induction, autophagosome formation, and autolysosome formation. Atg8-family proteins are valuable for tracking autophagic structures and have been widely utilized for monitoring autophagy. The conversion of LC3 to its lipidated form, LC3-II, served as an indicator of autophagy. Autophagy is implicated in human pathophysiology, such as neurodegeneration, cancer, and immune disorders. Moreover, autophagy impacts urological diseases, such as interstitial cystitis /bladder pain syndrome (IC/BPS), ketamine-induced ulcerative cystitis (KIC), chemotherapy-induced cystitis (CIC), radiation cystitis (RC), erectile dysfunction (ED), bladder outlet obstruction (BOO), prostate cancer, bladder cancer, renal cancer, testicular cancer, and penile cancer. Autophagy plays a dual role in the management of urologic diseases, and the identification of potential biomarkers associated with autophagy is a crucial step towards a deeper understanding of its role in these diseases. Methods for monitoring autophagy include TEM, Western blot, immunofluorescence, flow cytometry, and genetic tools. Autophagosome and autolysosome structures are discerned via TEM. Western blot, immunofluorescence, northern blot, and RT-PCR assess protein/mRNA levels. Luciferase assay tracks flux; GFP-LC3 transgenic mice aid study. Knockdown methods (miRNA and RNAi) offer insights. This article extensively examines autophagy's molecular mechanism, pharmacological regulation, and therapeutic application involvement in urological diseases.

Paradoxical Expression of R-10G-reactive Antigen in Human Testicular Embryonal Carcinoma.

Thus far, several monoclonal antibodies directed against cell-surface carbohydrate antigens have been generated. Among them, R-10G reportedly reacts selectively with human embryonic stem and induced pluripotent stem cells, but not with embryonal carcinoma (EC) cells. However, EC cells derived from patients' EC tumors may exhibit varying levels of R-10G-reactive antigen expression. Thus, we asked whether human EC tissues or germ cell tumor (GCT) tissues other than EC express R-10G-reactive antigen. To do so, we quantitatively analyzed R-10G-reactive antigen expression in 83 testicular GCT surgical specimens containing a total of 125 various GCT components. Accordingly, in all EC components examined, the EC cell plasma membrane was immunolabeled with R-10G, while most seminoma components were R-10G-negative. In non-seminomatous GCT (NSGCT) other than EC (non-EC NSGCT), R-10G-reactive antigen expression was variable, but signal distribution was focal, and the average intensity was weaker than that seen in EC. The percentages of R-10G-positive cells in these three groups varied with high statistical significance (p<0.001 for all combinations). These findings indicate that the R-10G-reactive antigen is preferentially expressed in human testicular EC tissues and, thus, could be used as a diagnostic marker for this malignancy.

The long non-coding RNA transcript, LOC100130460 (CAND1.11) gene, encodes a novel protein highly expressed in cancer cells and tumor human testis tissues.

BACKGROUND: Testis-specific genes encoding for long non-coding RNA (lncRNA) have been detected in several cancers; many produce proteins with restricted or aberrant expression patterns in normal or cancer tissues. OBJECTIVE: To characterize new lncRNA involved in normal and/or pathological differentiation of testicular cells. METHODS: Using bioinformatics analysis, we found that lncRNA LOC100130460 (CAND1.11) is expressed in normal and tumor testis; its expression was assessed in several human cell lines by qRT-PCR. CAND1.11 protein, produced by a single nucleotide mutation, was studied by western blot and immunofluorescence analysis on normal, classic seminoma, and Leydig cell tumor testicular tissues. RESULTS: CAND1.11 gene is primate-specific; its expression was low in SH-SY5Y cells and increased when differentiated with retinoic acid treatment. CAND1.11 expression in PC3 cells was higher than in PNT2 cells. CAND1.11 protein is present in the human testis and overexpressed in testicular cancer tissues. CONCLUSIONS: This report is one of the few providing evidence that a lncRNA produces a protein expressed in normal human tissues and overexpressed in several testicular cancers, suggesting its involvement in regulating cell proliferation and differentiation. Although further studies are needed to validate the results, our data indicate that CAND1.11 could be a potential new prognostic biomarker to use in proliferation and cancer.

Expression of Protein Markers in Spermatogenic and Supporting Sertoli Cells Affected by High Abdominal Temperature in Cryptorchidism Model Mice.

Cryptorchidism is a congenital abnormality resulting in increased rates of infertility and testicular cancer. We used cryptorchidism model mice that presented with the translocation of the left testis from the scrotum to the abdominal cavity. Mice underwent the surgical procedure of the left testis at day 0 and were sacrificed at days 3, 5, 7, 14, 21, and 28 post-operatively. The weight of the left cryptorchid testis decreased significantly at days 21 and 28. The morphological changes were observed after 5 days and showed detached spermatogenic cells and abnormal formation of acrosome at day 5, multinucleated giant cells at day 7, and atrophy of seminiferous tubules at days 21 and 28. The high abdominal temperature disrupted the normal expression of cell adhesion molecule-1, Nectin-2, and Nectin-3 which are essential for spermatogenesis. In addition, the pattern and alignment of acetylated tubulin in cryptorchid testes were also changed at days 5, 7, 14, 21, and 28. Ultrastructure of cryptorchid testes revealed giant cells that had been formed by spermatogonia, spermatocytes, and round and elongating spermatids. The study's findings reveal that cryptorchidism's duration is linked to abnormal changes in the testis, impacting protein marker expression in spermatogenic and Sertoli cells. These changes stem from the induction of high abdominal temperature.

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.

Myoid Gonadal Stromal Tumor: A New Case Report with a Review of the Literature.

The myoid gonadal stromal tumor is a very rare testicular spindle cell tumor. Few cases have been reported in the literature from 1977 to 2022. It was an emerging entity in the fourth edition of the WHO Classification of Tumors of the Urinary System and Male Genital Organs and has been instated as a full benign entity in the new edition of 2022. We report herein an additional case of a myoid gonadal stromal tumor. It was a 27-year-old man who presented with an asymptomatic mass in his left testicle for 2 months. The mass was confirmed by ultrasound as a 3 cm solid well-circumscribed lesion for which orchiectomy has been performed. The histological analysis of the specimen and the immunohistochemistry staining was consistent with a myoid gonadal stromal tumor. The characteristics of this tumor are summarized in well-circumscribed, nonencapsulated masses of uniform spindle cells, and immunohistochemically, tumor cells coexpress SMA and S-100 protein. The benign behavior of this tumor is demonstrated by our results and those presented in the previous cases reported in the literature.

Large cell calcifying Sertoli cell tumour: molecular and immunohistochemical assessment of a series comprising non-metastasising and metastasising neoplasms.

Large cell calcifying Sertoli cell tumour (LCCSCT) is a type of testicular sex cord-stromal tumour that may occur sporadically or in the context of Carney complex and other genetic syndromes. A subset is clinically malignant, and the molecular mechanisms that drive such aggressive behaviour remain unknown. METHODS AND RESULTS: We analysed 21 samples from 20 patients with LCCSCT (12 non-metastasising and eight metastasising) using PRKAR1A immunohistochemistry (IHC) and next-generation sequencing. All tumours except two (cases 17 and 20, both metastasising) demonstrated loss of PRKAR1A expression. Among 11 cases with interpretable sequencing results, all harboured pathogenic single nucleotide variants of PRKAR1A. Evidence of loss of heterozygosity (LOH) of PRKAR1A was present in all tumours with interpretable zygosity data, but the mechanisms of LOH were different for non-metastasising and metastasising tumours. Non-metastasising tumours demonstrated only copy-neutral LOH, while metastasising tumours demonstrated a spectrum of mechanisms of LOH, including copy-loss LOH, two concurrent mutations or copy-neutral LOH. Relevant molecular findings in non-metastasising LCCSCT were limited to PRKAR1A variants. In contrast, all metastasising LCCSCTs with interpretable data harboured additional pathogenic variants, including (but not restricted to) BRCA2 mutations with evidence of LOH and bi-allelic CDKN2A/B deletions. Three patients harboured PRKAR1A variants of inferred germline origin, including one with Carney complex and two without known syndromic features. CONCLUSIONS: This study further confirms that PRKAR1A IHC is a useful diagnostic tool for both non-metastasising and metastasising tumours and suggests that molecular analyses can be helpful to identify non-metastasising tumours with malignant potential in selected patients. Importantly, these results highlight that germline assessment could be beneficial for all patients presenting with LCCSCT.

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.

The seminal plasma microbiome of men with testicular germ cell tumours described by small RNA sequencing.

BACKGROUND: It has been estimated that microorganisms are involved in the pathogenesis of approximately 20% of all cancers. Testicular germ cell tumours (TGCTs) are the most common type of malignancy in young men and arise from the precursor cell, germ cell neoplasia in situ (GCNIS). The microbiome of seminal plasma and testicular tissue has not been thoroughly investigated in regard to TGCTs. OBJECTIVES: To investigate the differences in the seminal plasma microbiome between men with TGCT or GCNIS-only compared with controls. MATERIALS AND METHODS: The study population consisted of patients with GCNIS-only (n = 5), TGCT (n = 18), and controls (n = 25) with different levels of sperm counts in the ejaculate. RNA was isolated from the seminal plasma and sequenced. Reads not mapping to the human genome were aligned against a set of 2784 bacterial/archaeal and 4336 viral genomes using the Kraken pipeline. RESULTS: We identified reads from 2172 species and most counts were from Alteromonas mediterranea, Falconid herpesvirus 1, and Stigmatella aurantiaca. Six species (Acaryochloris marina, Halovirus HGTV-1, Thermaerobacter marianensis, Thioalkalivibrio sp. K90mix, Burkholderia sp. YI23, and Desulfurivibrio alkaliphilus) were found in significantly (q-value < 0.05) higher levels in the seminal plasma of TGCT and GCNIS-only patients compared with controls. In contrast, Streptomyces phage VWB was found at significantly higher levels among controls compared with TGCT and GCNIS-only patients combined. DISCUSSION: Often the microbiome is analysed by shotgun or 16S ribosomal sequencing whereas our present data build on small RNA sequencing. This allowed us to identify more viruses and phages compared with previous studies but also makes the results difficult to directly compare. CONCLUSION: Our study is the first to report identification of the microbiome species in seminal plasma of men with TGCT and GCNIS-only, which potentially could be involved in the pathogenesis of TGCTs. Further studies are, however, needed to confirm our findings.

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.