Development Of Testicular Germ Cell Tumors Research Articles

The association between Cannabis use and Testicular Germ Cell Tumor development

Since we are submitting a Letter to the Editor, we do not require an abstract.

Novel molecular aberrations involved in testicular germ cell tumor (TGCT) development and cisplatin resistance.

520 Background: Although highly curable, a proportion of testicular germ cell tumors (TGCT) develop resistance to cisplatin-based chemotherapy with subsequent disease relapses and possible patient death. The causes of disease progression and cisplatin (CDDP) resistance have not yet been well elucidated. Methods: We analyzed 56 samples of 15 patients with advanced and relapsing TGCT including primary tumors, metastases persisting after CDDP-containing chemotherapy, and circulating free tumor DNA (cfDNA) from the disease progression, with gDNA from blood leucocytes used as a background control. The quantity and quality of cfDNA was checked by capillary electrophoresis. All samples were sequenced by whole exome sequencing with SureSelect libraries on Illumina platform. Only mutations that were found in malignant samples or cfDNA with variant allele frequency (VAF) ≥ 5% and at least in 3 reads, not present in gDNA, with a minimum total read depth of 5 quality reads, excluding synonymous variants, were further analyzed. Results: Mutations of 6 genes with a significant role in carcinogenesis and/or testis development were identified in more than one patient: RBMX (in 4 pts), TPTE2 and ANKRD30A (in 3 pts), CDC27, PRAMEF8, and PRDM9 (in 2 pts). All the mutations were missense, with VAF 5 - 86%. They were all detectable in patients’ cfDNA, with increasing VAF during the tumor progression; the VAFs in cfDNA collected at advanced disease stages were even higher than those in primary tumors. PRDM9 mutations were found only in cfDNA from disease progression, but not in the primary testicular tumors. Of the identified genes, only CDC27 mutations have been previously described in TGCT. The genes newly associated with TGCT in this study are involved in genomic stability maintenance ( RBMX), spermiogenesis ( TPTE2), cell proliferation and apoptosis ( PRAMEF8), and several of them show testis-specific expression ( TPTE2, PRAMEF8, ANKRD30A, PRDM9). PRDM9 encodes a zinc-finger protein with histone methyltransferase activity, responsible for H3 methylation during meiosis. As epigenetic changes are supposed to play a crucial role in TGCT pathogenesis and resistance, and aberrant H3 methylation has been previously related to abnormal expression of OCT3/4 transcription factor promoting TGCT development, this gene is a strong candidate for TGCT progression and evolving CDDP resistance. Conclusions: Mutations of 5 novel genes have been identified in association with TGCT progression and cisplatin resistance. Their impact is strongly suggested by their functions in normal and malignant regulatory pathways and testis-specific expression patterns but has to be confirmed in experimental studies. The National Institute for Cancer Research project (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Supported by grants MH CZ - DRO (00064190, 00064203).

Subgrouping testicular germ cell tumors based on immunotherapy and chemotherapy associated lncRNAs

Testicular germ cell tumors (TGCT) are the most common reproductive system malignancies in men aged 15–44 years, accounting for 95 % of all testicular tumors. Our previous studies have been shown that long non-coding RNAs (lncRNAs), such as LINC00313, TTTY14 and RFPL3S, were associated with development of TGCT. Subgrouping TGCT according to differential expressed lncRNAs and immunological characteristics is helpful to comprehensively describe the characteristics of TGCT and implement precise treatment. In this study, the TGCT transcriptome data in The Cancer Genome Atlas Program (TCGA) database was used to perform consensus clustering analysis to construct a prognostic model for TGCT. TGCT was divided into 3 subtypes C1, C2, and C3 based on the differentially expressed lncRNAs. C1 subtype was sensitive to chemotherapy drugs, while the C2 subtype was not sensitive to chemotherapy drugs, and C3 subtype may benefit from immunotherapy. We defined the C1 subtype as epidermal progression subtype, the C2 subtype as mesenchymal progression subtype, and the C3 subtype as T cell activation subtype. Subgrouping based on differentially expressed genes (DEGs) and immunological characteristics is helpful for the precise treatment of TGCT.

Molecular Characterization of Patients with Cryptorchidism: Preliminary Search for an Expression Profile Related to That of Testicular Germ-Cell Tumors.

Cryptorchidism (CO) is a risk factor for the development of testicular germ-cell tumors (TGCT). This is supported by reports showing the persistence of gonocytes in CO patients. These cells are proposed to be related to the development of germ-cell neoplasia in situ (GCNIS), which is considered the precursor stage/lesion of TGCT. Therefore, it is proposed that some patients with CO could express some molecular markers related to TGCT. In this study, we analyzed testicular tissue samples from CO, TGCT, and controls. We determined the expression of POU5F1, PLAP, and KIT by immunohistochemistry and that of the hsa-miR-371-373 cluster, hsa-miR-367, and LATS2, PTEN, and IGFR1 genes by RT-qPCR. We then carried out a bioinformatic analysis to identify other possible candidate genes as tumor biomarkers. We found that 16.7% (2/12) of the CO patients presented increased expression of POU5F1, KIT, PLAP, hsa-miR-371-373, and hsa-miR-367 and decreased expression of LATS2 and IGF1R. Finally, the genes ARID4B, GALNT3, and KPNA6 were identified as other possible candidate tumor biomarkers. This is the first report describing the expression of the hsa-miR-371-373 cluster, hsa-miR-367, LATS2, and IGF1R in the testicular tissues of two CO patients with cells immune-positive to POU5F1, PLAP, and KIT, which is similar to what is observed in TGCT.

Prediagnostic Hormone Levels and Risk of Testicular Germ Cell Tumors: A Nested Case-Control Study in the Janus Serum Bank.

It has been hypothesized that poorly functioning Leydig and/or Sertoli cells of the testes, indicated by higher levels of serum gonadotropins and lower levels of androgens, are related to the development of testicular germ cell tumors (TGCT). To investigate this hypothesis, we conducted a nested case-control study within the Janus Serum Bank cohort. Men who developed TGCT (n = 182) were matched to men who did not (n = 364). Sex steroid hormones were measured using LC/MS. Sex hormone binding globulin, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were quantified by direct immunoassay. Multivariable logistic regression was used to calculate ORs and 95% confidence intervals (CI) for associations between hormone levels and TGCT risk. Higher FSH levels [tertile (T) 3 vs. T2: OR = 2.89, 95% CI = 1.83-4.57] were associated with TGCT risk, but higher LH levels were not (OR = 1.26, 95% CI = 0.81-1.96). The only sex steroid hormone associated with risk was androstane-3α, 17β-diol-3G (3α-diol-3G; OR = 2.37, 95% CI = 1.46-3.83). Analysis by histology found that increased FSH levels were related to seminoma (OR = 3.55, 95% CI = 2.12-5.95) but not nonseminoma (OR = 1.19, 95% CI = 0.38-3.13). Increased levels of 3α-diol-3G were related to seminoma (OR = 2.29, 95% CI = 1.35-3.89) and nonsignificantly related to nonseminoma (OR = 2.71, 95% CI = 0.82-8.92). Higher FSH levels are consistent with the hypothesis that poorly functioning Sertoli cells are related to the development of TGCT. In contrast, higher levels of 3α-diol-3G do not support the hypothesis that insufficient androgenicity is related to risk of TGCT. Clarifying the role of sex hormones in the development of TGCT may stimulate new research hypotheses.

The Polymorphisms in GSTO Genes (GSTO1 rs4925, GSTO2 rs156697, and GSTO2 rs2297235) Affect the Risk for Testicular Germ Cell Tumor Development: A Pilot Study.

Members of the omega class of glutathione transferases (GSTs), GSTO1, and GSTO2, catalyze a range of reduction reactions as a part of the antioxidant defense system. Polymorphisms of genes encoding antioxidant proteins and the resultant altered redox profile have already been associated with the increased risk for testicular germ cell cancer (GCT) development. The aim of this pilot study was to assess the individual, combined, haplotype, and cumulative effect of GSTO1rs4925, GSTO2rs156697, and GSTO2rs2297235 polymorphisms with the risk for testicular GCT development, in 88 patients and 96 matched controls, through logistic regression models. We found that carriers of the GSTO1*C/A*C/C genotype exhibited an increased risk for testicular GCT development. Significant association with increased risk of testicular GCT was observed in carriers of GSTO2rs2297235*A/G*G/G genotype, and in carriers of combined GSTO2rs156697*A/G*G/G and GSTO2rs2297235*A/G*G/G genotypes. Haplotype H7 (GSTO1rs4925*C/GSTO2rs2297235*G/GSTO2rs156697*G) exhibited higher risk of testicular GCT, however, without significant association (p > 0.05). Finally, 51% of testicular GCT patients were the carriers of all three risk-associated genotypes, with 2.5-fold increased cumulative risk. In conclusion, the results of this pilot study suggest that GSTO polymorphisms might affect the protective antioxidant activity of GSTO isoenzymes, therefore predisposing susceptible individuals toward higher risk for testicular GCT development.

Silencing of LINC00467 inhibits cell proliferation in testicular germ cell tumors cells.

A significant decrease in LINC00467 expression in testicular germ cell tumors (TGCTs) was found in our previous study in comparison to adjacent tissue. Interestingly, the expression of LINC00467 correlated with the pathological grade of the tumor in TGCT patients. The higher the expression of LINC00467 was, the worse the prognosis of the patients with TGCT was. Despite these findings, the exact role of LINC00467 in the development of TGCTs requires further investigation. LINC00467 expression was downregulated in the NCCIT and TCam-2 cell lines via small interfering RNA (siRNA) silencing. The levels of gene expression were validated using quantitative real-time polymerase chain reaction (qRT-PCR) analyses. Cell proliferation was evaluated by the MTT and Cell Counting Kit-8(CCK8) assays, whereas flow cytometry was used to assess the effects on the cell cycle. Western blotting analysis was used to detect expression levels of protein. Additionally, RNA-sequencing and bioinformatics methods were used to investigate the mechanism of action of LINC00467 in TGCTs. The suppression of LINC00467 expression resulted in decreased cell proliferation and induced S-phase arrest. Furthermore, the suppression of LINC00467 downregulated proliferating cell nuclear antigen (PCNA), a protein related to cell cycle regulation, while it upregulated p21 expression. In other studies involving dihydrotestosterone (DHT) stimulation, it was observed that DHT could upregulate LINC00467 expression. In addition, silencing of the LINC00467 reversed the effect of testosterone on cell proliferation. The Gene Set Enrichment Analysis (GSEA) revealed that LINC00467 regulated the p53 pathway by modulating the expression of CCNG1. Our study found that LINC00467 regulates cell proliferation by inducing S-phase arrest through the cell cycle-related proteins PCNA and p21. These findings contribute to our understanding of non-coding RNAs mechanisms involved in the development of TGCTs.

Paternal Occupational Exposure to Heavy Metals and Welding Fumes and Testicular Germ Cell Tumours in Sons in France.

Simple SummaryTesticular cancer is the most common cancer among men below 40 years old, and its causes remain largely unknown. Marked geographic differences in the occurrence along with the relatively young age at diagnosis suggest a possible role of environmental and occupational exposures that occur early in life, probably already during embryonic development. Using data from a French case–control study, we investigated associations between paternal occupational exposure to five heavy metals and welding fumes before and at birth and testicular cancer risk in sons. We estimated exposures from job titles and considered other potential risk factors in the statistical analyses. We found no association. Thus, further research is necessary to identify potentially modifiable risk factors.Testicular cancer is the most common cancer in young men. Its causes are largely unknown, although prenatal occupational and environmental exposures have been suggested. We investigated paternal occupational exposure to heavy metals and welding fumes and the risk of testicular germ cell tumors (TGCT) in their offspring. A total of 454 cases and 670 controls were included from a French nationwide case–control study. The INTEROCC job exposure matrix was used to assign occupational exposures (cadmium, chromium, iron, nickel, lead, and welding fumes) to the fathers’ jobs. Odds ratios (ORs) for TGCT were estimated using conditional logistic regression models for frequency-matched sets. Three complementary analytical approaches were used: (1) single-agent analysis, (2) analysis by groups, and (3) principal component analysis (PCA). The proportion of paternal exposure to different heavy metals and welding fumes ranged from 0.7% (cadmium) to 11.3% (lead). Based on PCA, three principal components explained 93.5% of the cumulative variance. No associations were found between heavy metals or welding fumes and TGCT. In this study, paternal occupational exposure to heavy metals or welding fumes was not associated with TGCT development in their sons.

A Role of the TEX101 Interactome in the Common Aetiology Behind Male Subfertility and Testicular Germ Cell Tumor.

Patients who develop testicular germ cell tumours (TGCT) are at higher risk to be subfertile than the general population. The conditions are believed to originate during foetal life, however, the mechanisms behind a common aetiology of TGCT and male subfertility remains unknown. Testis-expressed 101 (TEX101) is a glycoprotein that is related to male fertility, and downregulation of the TEX101 gene was shown in pre-diagnostic TGCT patients. In this review, we summarize the current knowledge of TEX101 and its interactome related to fertility and TGCT development. We searched literature and compilation of data from curated databases. There are studies from both human and animals showing that disruption of TEX101 result in abnormal semen parameters and sperm function. Members of the TEX101 interactome, like SPATA19, Ly6k, PICK1, and ODF genes are important for normal sperm function. We found only two studies of TEX101 related to TGCT, however, several genes in its interactome may be associated with TGCT development, such as PLAUR, PRSS21, CD109, and ALP1. Some of the interactome members are related to both fertility and cancer. Of special interest is the presence of the glycosylphosphatidylinositol anchored proteins TEX101 and PRSS21 in basophils that may be coupled to the immune response preventing further development of TGCT precursor cells. The findings of this review indicate that members of the TEX101 interactome could be a part of the link between TGCT and male subfertility.

Diverse Roles and Targets of miRNA in the Pathogenesis of Testicular Germ Cell Tumour.

Simple SummaryTesticular germ cell tumour (TGCT) is the most common malignancy among young males in many parts of the world. Although it is highly remediable, treatments lead to long-term comorbidities, and therefore, it warrants a better prognosis. The presence of several risk loci in non-coding regions supports a functional role of miRNAs in TGCT development, and recent studies point to the emerging roles of miRNA and the complexity of miRNA-mediated gene regulation in TGCTs. miRNAs may act as oncogenes or tumour suppressors in TGCT by regulating targets involved in cell proliferation, apoptosis, and metastasis. Here, we summarise the gene regulation and function of miRNAs involved in TGCT pathogenesis.Testicular germ cell tumour (TGCT) is the most common cancer type among young adults in many parts of the world. Although the pathogenesis of TGCT is not well understood, the involvement of heritable components is evident, and the risk is polygenic. Genome-wide association studies have so far found 78 susceptibility loci for TGCT, and many of the loci are in non-coding regions indicating the involvement of non-coding RNAs in TGCT pathogenesis. MicroRNAs (miRNAs), a class of non-coding RNAs, have emerged as important gene regulators at the post-transcriptional level. They are crucial in controlling many cellular processes, such as proliferation, differentiation, and apoptosis, and an aberrant miRNA expression may contribute to the pathogenesis of several cancers, including TGCT. In support of this notion, several studies reported differential expression of miRNAs in TGCTs. We previously demonstrated that miRNAs were the most common group of small non-coding RNAs in TGCTs, and several functional studies of miRNAs in TGCTs suggest that they may act as either oncogene or tumour suppressors. Moreover, individual miRNA targets and downstream pathways in the context of TGCT development have been explored. In this review, we will focus on the diverse roles and targets of miRNAs in TGCT pathogenesis.

Role of Hub Genes in the Occurrence and Development of Testicular Cancer Based on Bioinformatics.

BackgroundTesticular cancer severely affects male health, so finding effective diagnosis and prognostic indicators and exploring its pathogenesis are very important.PurposeThis study aims to explore the hub genes that play important roles in the occurrence and development of testicular germ cell tumor (TGCT).MethodsData were obtained from Gene Expression Omnibus datasets (GSE3218 and GSE1818) and verified in The Cancer Genome Atlas database and the Genotype-Tissue Expression database and the Human Protein Atlas database. A protein–protein interaction network was constructed to obtain hub genes. GEO2R, R software and packages were used to analyze differentially expressed genes (DEGs), receiver operating characteristic curve assessment, Cox regression analysis, Kaplan–Meier survival curve assessment, Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, the relationship with clinicopathological information, gene set enrichment analysis, the correlation with immune cells’ infiltration, and the expression in pan-cancers of the hub genes.ResultsPLK4, TRIP13, TPR, KIF18A, CDKN3, HMMR, PBK, PTTG1, CKS2, SYCP1, HSPA2, and MKI67 were selected as the hub genes. mRNA of PLK4, TRIP13, CDKN3, SYCP1, HSPA2, and MKI67 had high diagnostic values, and higher expression of CDKN3 and HSPA2 mRNA were poor prognostic factors for progression-free interval of TGCT. The hub genes involved organelle division and cell cycle, chromosome and centromeric region, heat shock protein binding, and more. Downregulated TPR and PLK4 were selected as research targets for continued study, and they may participate in multiple signaling pathways. The expression of TPR and PLK4 correlated with the infiltration of a variety of immune cells and differed in pan-cancers.ConclusionThe mRNA levels of multiple hub genes have high diagnostic and prognostic values for TGCT. TPR and PLK4 may play a role in the occurrence and development of TGCT through cancer-related signaling pathways.

Identification of new biomarkers in immune microenvironment of testicular germ cell tumour.

To seek novel prognostic biomarkers for testicular germ cell tumour (TGCT) and investigate the tumour immune microenvironment, we identified critical differentially expressed genes (DEGs) by overlapping GSE1818 dataset from Gene Expression Omnibus (GEO). Protein-protein interaction (PPI) network was used to investigate key modules and hub genes. Functional enrichment analysis was performed to investigate the underlying molecular functions of the DEGs in TGCT development and progression. The following survival analysis based on The Cancer Genome Atlas (TCGA) TGCT dataset indicated that AKAP4, SPA17 and TNP1 are correlated with TGCT prognosis. Immunohistochemistry and quantitative real-time polymerase chain reaction verified the down-regulation of the 3 hub genes in TGCT. Gene set enrichment analysis was conducted to further explore the role of the 3 hub genes in TGCT respectively. In addition, TGCT samples had high infiltration of CD8+ T cells, M0 and M1 macrophage cells, and resting myeloid dendritic cells in immune microenvironment. We also constructed the microRNA-gene regulatory networks to identify the key upstream microRNAs in TGCT. In conclusion, our findings indicated that AKAP4, SPA17 and TNP1 are promising biomarkers of TGCT. AKAP4 and TNP1 might regulate immune cells infiltration in immune microenvironment.

Serum RNA Profiling in the 10-Years Period Prior to Diagnosis of Testicular Germ Cell Tumor.

Although testicular germ cell tumor (TGCT) overall is highly curable, patients may experience late effects after treatment. An increased understanding of the mechanisms behind the development of TGCT may pave the way for better outcome for patients. To elucidate molecular changes prior to TGCT diagnosis we sequenced small RNAs in serum from 69 patients who were later diagnosed with TGCT and 111 matched controls. The deep RNA profiles, with on average 18 million sequences per sample, comprised of nine classes of RNA, including microRNA. We found that circulating RNA signals differed significantly between cases and controls regardless of time to diagnosis. Different levels of TSIX related to X-chromosome inactivation and TEX101 involved in spermatozoa production are among the interesting findings. The RNA signals differed between seminoma and non-seminoma TGCT subtypes, with seminoma cases showing lower levels of RNAs and non-seminoma cases showing higher levels of RNAs, compared with controls. The differentially expressed RNAs were typically associated with cancer related pathways. Our results indicate that circulating RNA profiles change during TGCT development according to histology and may be useful for early detection of this tumor type.

An update on microRNAs as potential novel therapeutic targets in testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) are the most frequent solid malignant tumors in men 20- 40 years of age and the most frequent cause of death from solid tumors in this age group. Recent studies have underscored the fact that miRNA deregulation is a feature of carcinogenesis, including TGCT development and progression. MiRNAs are a group of small noncoding RNAs that bind to the 3'-untranslated region (UTR) of the targeted mRNAs, thus causing mRNA degradation or the inhibition of its translation, regulating gene expression in a temporal and tissue-specific manner. However, few miRNAs have been found to play key roles in TGCTs; recently, other miRNAs have been identified, representing novel potential therapeutic targets.

Cryptorchidism and Testicular Tumor: Comprehensive Analysis of Common Clinical Features and Search of SNVs in the KIT and AR Genes

Allelic variants in genes implicated in the development of testicular germ cell tumor (TGCT) could be present in patients with cryptorchidism (CO). Currently; the mechanisms explaining this relationship are still unknown. In this study the common clinical features in patients with CO and TGCT and 6 variants of KIT and AR genes associated to TGCT were analyzed. Population analyzed included 328 individuals: 91 patients with CO; 79 with TGCT, 13 of them with previous CO diagnosis, and 158 healthy males. Of the 13 patients with TGCT and history of CO, one patient (7.7%) presented the heterozygous form of the variant rs121913507 and two patients (15.4%) presented homozygote genotype for the variant rs121913506 in KIT gene. Interestingly, the heterozygous form for the variant rs121913506 of KIT gene was identifying in all of 13 patients. The rs201934623, rs774171864, and rs12014709 variants of the AR gene did not show any clinical association. Our results strongly support that genetic component in CO could be conditioning for the development of TGCT. Notably, KIT gene variants might be determinants in the pathological association between TGCT and CO.

Short anogenital distance is associated with testicular germ cell tumour development.

Testicular germ cell tumour is a multifactorial disease in which various genetic and environmental factors play a role. Testicular germ cell tumour is part of the testicular dysgenesis syndrome which includes also cryptorchidism, hypospadias, oligo/azoospermia and short anogenital distance. The primary objective was to examine anogenital distance in testicular germ cell tumour cases and healthy fertile controls. The secondary objective was to assess the (CAG)n polymorphism of the Androgen Receptor gene in relationship with anogenital distances and testicular germ cell tumour development. 156 testicular germ cell tumour patients and 110 tumour-free normozoospermic controls of Spanish origin. All subjects underwent full andrological workup (including semen and hormone analysis) and genetic analysis (Androgen Receptor (CAG)n). The main outcome measures were the anopenile distance (AGDap), the anoscrotal distance (AGDas) and AR(CAG)n. We observed significantly shorter anogenital distances in the group of testicular germ cell tumour patients in respect to controls (P<.001) independently from sperm count and testis histology. Threshold values, applicable only to our cohort, were calculated for anogenital distances with the best sensitivity and specificity. Subjects with AGDap and AGDas below threshold showed a significantly increased risk for testicular germ cell tumour (OR=4.97, 95% CI=2.01-12.33, P=.001 and OR=4.11, 95% CI=1.89-8.92, P≤.001, respectively). No significant correlation was observed between AR(CAG)n polymorphism and anogenital distances. The median values of the AR(CAG)n were similar between cases and controls, excluding a major role for this polymorphism in the etiopathogenesis of these testicular dysgenesis syndrome components. Ours is the first study focusing on anogenital distances in testicular germ cell tumour patients. We identified short anogenital distances (which is a surrogate biomarker of androgen action during foetal life) as a significant risk factor for this disease. After further validation of our preliminary data, anogenital distance measurement could become part of testicular germ cell tumour screening in order to better define those individuals who would benefit from long-term active follow-up.

SPANXN2 functions a cell migration inhibitor in testicular germ cell tumor cells.

BackgroundSPANX family members are thought to play an important role in cancer progression. The SPANXN2 is a gene expressed mainly in normal testis, but its role in testicular germ cell tumors (TGCTs) has yet to be investigated. TGCT is one of the most common solid tumors in young men and is associated with poor prognosis; however, effective prognostic indicators remain elusive. Therefore, we investigated the role of SPANXN2 in TGCT development.MethodsSPANXN2 expression levels were validated by quantitative real-time polymerase chain reaction (qRT-PCR) analyses of 14 TGCT samples and five adjacent normal tissue samples. SPANXN2 was transiently overexpressed in TGCT cells to study the consequences for cell function. The effects of SPANXN2 on cell migration were evaluated in transwell and wound healing assays. The effects on cloning ability were evaluated in colony formation assays. MTT assays and cell cycle analysis were used to detect the effects of SPANXN2 on cell proliferation. The expression levels of EMT- and AKT-related proteins in cells overexpressing SPANXN2 were analyzed by Western blotting.ResultsCompared with adjacent normal tissues, the Gene Expression Profiling Interactive Analysis database showed SPANXN2 expression was downregulated in TGCTs which was consistent with the qRT-PCR analysis. SPANXN2 overexpression reduced cell migration and colony formation capability and downregulated expression of EMT- and AKT-related proteins, Vimentin, Snail, AKT, and p-AKT.ConclusionOur results suggest that SPANXN2 regulates TGCT cell migration via EMT- and AKT-related proteins although its role in the occurrence and development of TGCT remains to be fully elucidated.

The impact of circulating free tumor DNA (cfDNA) in testicular germ cell tumors (TGCT) management.

e17519 Background: The clinical management of testicular germ cell tumors (TGCT) has not changed much for decades; most importantly, novel prognostic factors indicating the need of adjuvant chemotherapy and factors related to the development of cisplatin resistance would be needed. Circulating free tumor DNA (cfDNA) is an easily available and valuable source of genetic material, which may bring clinically relevant information. Methods: After ethical committee approval and patient informed consent, peripheral blood (PB) samples were taken from TGCT patients at the diagnosis (after orchidectomy), after 2 cycles of chemotherapy, at the end of treatment, and at relapse or disease progression, if applicable. Clinical data of all patients were recorded. The PB samples were processed immediately after collection, plasma was separated by centrifugation, cfDNA was extracted by QIAmp Circulating Nucleic Acid kits (Qiagen), its quality and quantity was assessed by capillary electrophoresis (Agilent) and qPCR for a house-keeping gene. Selected samples were subjected to whole exome sequencing using SureSelectXT HS + Human All Exon v6 kits (Agilent) on NextSeq (Illumina) platform, together with the corresponding primary testicular tumor and peripheral blood mononuclears as a germ-line control. Statistical analyses were performed using non-parametric tests. Results: Sixty-three samples of 41 patients have been analyzed. The median amount of detected cfDNA did not significantly differ from non-malignant controls, was similar in seminoma and non-seminoma pts, but was significantly higher (p = 0.01) in pts with disease progression. In pts with elevated cfDNA levels, these decreased after 2 and 4 cycles of chemotherapy. In 5 sequenced pts, molecular aberrations (somatic missense or frameshift mutations) were found in genes CDC27 (2 pts), RBMX (4 pts), TPTE2 (3 pts), and TSPAN16 (1 pt). These aberrations were also detected in primary tumors but with lower frequencies, and were not present in germ-line DNA. CDC27 mutations have been described in TGCT previously. The other genes have not yet been linked to TGCT, although their role in spermatogenesis and cell proliferation is well known. The presence of mitochondrial DNA was not detected in cfDNA. Conclusions: High levels of cfDNA are detectable in pts with disease progression where they reflect the total tumor load; the molecular analysis of cfDNA revealed novel aberrations that may play a role in TGCT development. Supported by grants MH CZ - DRO00064190TN, CDRO00064203FNM and MEYS NPU I LO1604

METTL3 May Regulate Testicular Germ Cell Tumors Through EMT and Immune Pathways

Testicular germ cell tumors (TGCTs) are highly prevalent in young men aged 20–40 years and are one of the most common lethal solid tumors in men of this age. Due to the current unclear mechanism of tumor development, there is a lack of effective treatment, and therefore in-depth research of the molecular mechanism of the occurrence and development of TGCT and the search for suitable and effective therapeutic targets and molecular markers are of great significance for achieving effective treatment. METTL3 is a very important methylase, which has been implicated in the progression of many cancers, but the role of METTL3 in TGCT has not been fully elucidated. In this article, we found that METTL3 expression was significantly downregulated in TGCT tissues, and patients with low expression levels had lower overall survival and relapse-free survival rates. After overexpressing METTL3, cell proliferation, invasion, and migration ability significantly increased, while influencing the expression of epithelial–mesenchymal transition (EMT)-related proteins. In addition, we observed that the expression level of METTL3 positively correlated with molecular markers and infiltration level of CD8+ and CD4+ T cells and natural killer cells. In sum, our findings identified that METTL3 can be used as an independent prognostic marker in patients with TGCT. METTL3 participates in the proliferation, migration, and invasion of TGCT cells by regulating the expression of EMT-related genes and may also play a role in activating the tumor immune response in TGCT.

Molecular Basis of Cisplatin Resistance in Testicular Germ Cell Tumors

The emergence of cisplatin (CDDP) resistance is the main cause of treatment failure and death in patients with testicular germ cell tumors (TGCT), but its biologic background is poorly understood. To study the molecular basis of CDDP resistance in TGCT we prepared and sequenced CDDP-exposed TGCT cell lines as well as 31 primary patients’ samples. Long-term exposure to CDDP increased the CDDP resistance 10 times in the NCCIT cell line, while no major resistance was achieved in Tera-2. Development of CDDP resistance was accompanied by changes in the cell cycle (increase in G1 and decrease in S-fraction), increased number of acquired mutations, of which 3 were present within ATRX gene, as well as changes in gene expression pattern. Copy number variation analysis showed, apart from obligatory gain of 12p, several other large-scale gains (chr 1, 17, 20, 21) and losses (chr X), with additional more CNVs found in CDDP-resistant cells (e.g., further losses on chr 1, 4, 18, and gain on chr 8). In the patients’ samples, those who developed CDDP resistance and died of TGCT (2/31) showed high numbers of acquired aberrations, both SNPs and CNVs, and harbored mutations in genes potentially relevant to TGCT development (e.g., TRERF1, TFAP2C in one patient, MAP2K1 and NSD1 in another one). Among all primary tumor samples, the most commonly mutated gene was NSD1, affected in 9/31 patients. This gene encoding histone methyl transferase was also downregulated and identified among the 50 most differentially expressed genes in CDDP-resistant NCCIT cell line. Interestingly, 2/31 TGCT patients harbored mutations in the ATRX gene encoding a chromatin modifier that has been shown to have a critical function in sexual differentiation. Our research newly highlights its probable involvement also in testicular tumors. Both findings support the emerging role of altered epigenetic gene regulation in TGCT and CDDP resistance development.