UNASSIGNED: Lymphovascular invasion (LVI) is a high-risk factor for testicular germ-cell tumors (TGCT), but a prognostic model for TGCT-LVI patients is lacking. This study aimed to develop a nomogram for predicting the overall survival (OS) of TGCT-LVI patients.
UNASSIGNED: A complete cohort of 3288 eligible TGCG-LVI patients (training cohort, 2300 cases; validation cohort, 988 cases) were obtained from the Surveillance, Epidemiology, and End Results database. Variables screened by multivariate Cox regression analysis were used to construct a nomogram, which was subsequently evaluated using the consistency index (C-index), time-dependent receiver operating characteristic curve (ROC), and calibration plots. The advantages and disadvantages of the American Joint Committee on Cancer (AJCC) staging system and the nomogram were assessed by integrated discrimination improvement (IDI) and net reclassification improvement (NRI). Decision-analysis curve (DCA) was used to measure the net clinical benefit of the nomogram versus the AJCC staging system. Finally, Kaplan-Meier curves were used to evaluate the ability to identify different risk groups between the traditional AJCC staging system and the new risk-stratification system built on the nomogram.
UNASSIGNED: Nine variables were screened by multivariate Cox regression analysis to construct the nomogram. The C-index (training cohort, 0.821; validation cohort, 0.819) and time-dependent ROC of 3-, 5-, and 9-year OS between the two cohorts suggested that the nomogram had good discriminatory ability. Calibration curves showed good consistency of the nomogram. The NRI values of 3-, 5-, and 9-year OS were 0.308, 0.274, and 0.295, respectively, and the corresponding values for the validation cohort were 0.093, 0.093, and 0.099, respectively (P<0.01). Additionally, the nomogram had more net clinical benefit as shown by the DCA curves, and the new risk-stratification system provided better differentiation than the AJCC staging system.
UNASSIGNED: A prognostic nomogram and new risk-stratification system were developed and validated to assist clinicians in assessing TGCT-LVI patients.

OBJECTIVE: With the heterogeneous genetic background, prognosis prediction and therapeutic targets for testicular germ cell tumors (TGCTs) are still unclear. We defined the tumor immune microenvironment activation status (TIMEAS).
METHODS: We collected a total of 314 TGCT patients from four cohorts, including a 48-case microarray. A nonnegative matrix factorization algorithm was applied to identify the \"immune factor\", derived the top 150 weighted genes to divide patients into immune and non-immune classes, and further separated the immune class into activated and exhausted subgroups by nearest template prediction. Tumor mutant burden, gene mutation, and copy number alteration were compared with our recently developed package \"MOVICS\". A random forest algorithm was performed to establish a prediction model with fewer genes. Immunohistochemistry staining was performed to identify TIMEAS in the microarray.
RESULTS: We constructed the TIMEAS in the TCGA-TGCT cohort and further validated it in the GSE3218 and GSE99420 cohorts. The immune class contained the activated status of T-lymphocytes, B-lymphocytes, and macrophages, while Treg cells and the WNT/TGFβ signature were more activated in the immune-suppressed subgroup. Patients in the immune-exhausted subgroup had the worst prognosis, and 22.9% of patients in the immune-activated subgroup had KRAS mutations, which might stimulate the response of the immune system and lead to a favorable prognosis. The immune-exhausted group benefited more from chemotherapy, while the immune-activated subgroup responded well to anti-PD-1/PD-L1 therapy. FSCN1 was validated as the target of the immune-exhausted microenvironment by immunohistochemistry.
CONCLUSIONS: TIMEAS classification can separate TGCT patients; patients in the immune-activated subgroup could benefit more from anti-PD-L1 immunotherapy, and those in the immune-exhausted subgroup are more suitable for chemotherapy.

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

BACKGROUND: Accumulating evidence has indicated that aberrant RNA modifications are associated with malignant progression and the immune microenvironment in various tumors. However, the function of RNA modification regulators in testicular germ cell tumors (TGCTs) remains to be discovered. This study aimed to investigate the biological functions of RNA modification regulators in testicular germ cell tumors and identify their potential clinical predictive value.
METHODS: Expression level of 75 RNA modification regulators was acquired to generate differential expression patterns. RNA modification regulatory genes were applied to construct a progression-free survival (PFS) risk model. Meanwhile, three RNA modification clusters were identified using consensus clustering. Subsequently, the infiltration characteristics of cells in the microenvironment as well as the antitumor drug candidates have been further analyzed. Finally, to further validate our results, we examined the expression and biological behavior of seven selected RNA modification regulators both in TGCT cell lines and clinical tissues.
RESULTS: We collected the differentially expressed regulators of RNA modification. RNA modification risk signature was developed to stratify the prognosis of TGCT patients. Furthermore, we found significant differences in immune microenvironment between subgroups. Ultimately, seven selected RNA modification regulators were further verified.
CONCLUSIONS: We generated and validated a risk signature related to RNA modification which could accurately predict the relapse risk in TGCT patients. This risk signature was correlated with immune cells infiltration among tumor microenvironments. Furthermore, we screened antitumor drug candidates and evaluated the sensitivity and efficacy of class chemotherapeutic drugs, which could provide reference for clinical drug use.

BACKGROUND: Stem cell niche maintains stem cell population identity and is essential for the homeostasis of self-renewal and differentiation in Drosophila testes. However, the mechanisms of CySC lineage signals-mediated soma-germline communications in response to external stimuli are unclear.
METHODS: Pre-initiation complex functions were evaluated by UAS-Gal4-mediated cell effects. RNA sequencing was conducted in NC and eIF5 siRNA-treated cells. Genetic interaction analysis was used to indicate the relationships between eIF5 and eIF1A/eIF2γ in Drosophila testes.
RESULTS: Here, we demonstrated that in CySCs, translation initiation factor eIF5 mediates cyst cell differentiation and the non-autonomously affected germ cell differentiation process. CySCs lacking eIF5 displayed unbalanced cell proliferation and apoptosis, forming testicular germ cell tumors (TGCTs) during spermatogenesis. eIF5 transcriptional regulation network analysis identified multiple metabolic processes and several key factors that might be involved in germ cell differentiation and TGCT formation. Importantly, knockdown of eIF1A and eIF2γ, key components of pre-initiation complex, mimicked the phenotype of knocking down eIF5 in the stem cell niche of Drosophila testes. Genetic interaction analysis indicated that eIF5 was sufficient to rescue the phenotype of tumorlike structures induced by down-regulating eIF1A or eIF2γ in CySCs.
CONCLUSIONS: These findings demonstrated that CySC lineage eIF5, together with eIF1A or eIF2γ, mediates soma-germline communications for the stem cell niche homeostasis in Drosophila testes, providing new insights for the prevention of TGCTs.

The Kelch-like protein 11 antibody-associated paraneoplastic neurological syndrome (KLHL 11-PNS) was first identified in 2019. This novel antibody, targeting the intracellular KLHL 11 antigen, can be detected in serum and cerebrospinal fluid using tissue-based and cell-based assays. It is thought to be a biomarker for a T-cell autoimmunity response. The most likely immunopathogenesis of KLHL 11-PNS appears to be linked to cytotoxic T-cell-mediated neuronal injury and loss. Patients have adult-male predilection, rhombencephalitis (brainstem and / or cerebellar involvement), and a robust oncological correlation with testicular germ cell tumors (predominately seminoma). Brain magnetic resonance imaging demonstrated T2 / fluid-attenuated inversion recovery hyperintensities and atrophy of the temporal lobe, cerebellum, and brainstem. Most patients responded poorly to immunotherapy and oncotherapy and thus had a poor long-term prognosis. We review the literature and provide an update of current knowledge regarding KLHL 11-PNS, including epidemiology, underlying mechanism, clinical presentations, paraclinical and oncological findings, diagnostic workup, and treatment approaches.

The incidence of testicular germ cell tumor (TGCT) is currently on the rise worldwide, of which 15%-30% of patients have occur recurrence and metastasis. However, clinical methods for diagnosing TGCT and judging its prognosis remained inadequate. In this study, we aimed to explore the possibility of testis-specific long-chain non-coding RNA (lncRNA) Ret finger protein-like 3S (RFPL3S) as a biomarker for TGCT diagnosis, prognosis, and treatment response by reviewing the TGCT gene expression data in Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. The cohort data and DNA methylation data of TGCT in TCGA were downloaded from TGCA, UCSC XENA, and GEO. The bioinformatic tools were used, including GEPIA2, Kaplan-Meier Plotter, LinkedOmics, UCSC XENA, Sangerbox Tools, GSCA, and Tumor Immune Dysfunction and Exclusion. Compared with normal testicular tissues, the RFPL3S expression was significantly reduced in TGCT, and was significantly negatively correlated with the patient\'s Tumor, Node, Metastasis stage. Hypermethylation and low copy number of RFPL3S were present in TGCT, and low RFPL3S was associated with short disease-free and progression-free intervals. Silencing RFPL3S significantly enhanced the invasion ability and proliferation ability of TGCT cells as evaluated by Transwell and CCK-8 experiments. Additionally, RFPL3S expression was positively correlated with the infiltration of immune-activating cells such as B cells, CD8+ T cells, cytotoxic T cells, and natural killer cells, and negatively correlated with the infiltration of immunosuppressive cells such as Th17 and Th2. Higher RFPL3S expression was present in patients with immunotherapy benefits. In conclusion, we determined that the testis-specific lncRNA RFPL3S functioned as a tumor suppressor in TGCT and could be used as a prognostic predictor of TGCT, as well as a marker to predict the effect of TGCT immunotherapy.

Testicular germ cell tumor (TGCT) is a relatively rare entity tumor, accounting for only 1% of all male cancers. However, it is the most common solid tumor in young men between 15 and 34 years old. Long noncoding RNAs (lncRNAs) are involved in various physiological and pathological processes. However, the functions of lncRNAs in TGCT have only rarely been investigated. LncRNAs associated with TGCT were identified using Gene Expression Omnibus (GEO) database and UCSC XENA database data mining. The effects of LINC00313 on NCCIT cell migration and invasion were evaluated in transwell assays. The expression levels of epithelial-mesenchyme transition (EMT)-related proteins in cells knockdown of LINC00313 were analyzed by Western blot. Correlation analyses between lncRNA LINC00313 expression and copy number variation (CNV) and immune cell infiltration were carried out using The Cancer Genome Atl as (TCGA) data. The effect of Panobinostatin targeting LINC00313 in TGCT cells was investigated. We observed higher LINC00313 expression in TGCT. The migratory and invasive properties of TGCT cells were augmented by LINC00313, likely via its effects on modulating the expression of epithelial-mesenchyme transition (EMT) related proteins: CTNNB1, ZEB1, CDH2, Snail and VIM. Moreover, LINC00313 expression and CNV correlated negatively with the infiltration of immune cells. In addition, Panobinostat might be a possible candidate drug to target LINC00313 in TGCT. LINC00313 performs important pro-migration and invasion functions in the pathogenesis of TGCT. LINC00313 could be used as diagnostic, prognostic, immune marker and therapeutic target to develop effective treatment of TGCT.

Embryonal carcinoma (EC) and seminoma (SE) are both derived from germ cell neoplasia in situ but show big differences in growth patterns and clinical prognosis. Epigenetic regulation may play an important role in the development of EC and SE. This study investigated the DNA methylation-based genetic alterations between EC and SE by analyzing the datasets of mRNA expression and DNA methylation profiling. The datasets were downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified between EC and SE by limma package in R environment. Gene function enrichment analysis of the DEGs was performed on the DAVID tool, the results of which suggested differences in capability of pluripotency and genomic stability between EC and SE. The minfi package and wANNOVAR tool were used to identify differentially methylated genes. A total of 37 genes were discovered with both mRNA expression and the accordant DNA methylation changes. The findings were verified by the sequencing data from The Cancer Genome Atlas database, and Kaplan-Meier survival analysis was performed. Finally, 5 genes (PRDM1, LMO2, FAM53B, HCN4, and FAM124B) were found that showed both low expression and high methylation in EC, and were significantly associated with relapse-free survival. The findings of methylation-based genetic features between EC and SE might be helpful in studying the role of DNA methylation in cancer development.

In a population-based case control study of testicular germ cell tumors (TGCT), we reported a strong positive association between serum levels of Wolff\'s Group 1 (potentially estrogenic) polychlorinated biphenyl (PCBs) and risk of TGCT, and the observed associations were similar for both seminoma and non-seminoma. While the observed specific associations between TGCT and Wolff\'s Group 1 PCBs cannot be easily explained by bias or confounding, a question can still be asked, that is, could the relationship between PCBs and TGCT differ by age at diagnosis? PCBs tend to bioaccumulate, with more heavily chlorinated PCB congeners tending to have longer half-lives. Half-lives of PCB congeners were reported ranging from 4.6 years for PCB-28 to 41.0 years for PCB-156. The half-life for the heavy PCB congeners (17.8 years) was found to be approximately twice that for the light PCBs (9.6 years) in early studies. Therefore, the same PCB concentration measured in a 20-year-old vs. a 55-year-old is unlikely to represent the same lifetime PCB exposure or type of PCB exposure. In this analysis, we stratified the data by median age of diagnosis of TGCT and further stratified by histologic type of TGCT (seminoma vs non-seminoma) to explore if the risk of TGCT associated with PCB exposures differs by age.