Development Of 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.

P-606 Maintenance of telomere length through autophagy and androgen receptor inhibition in infertile individuals with differences of sex development (DSD)

Abstract Study question What are the molecular mechanisms underlying genome instability concomitant with increased telomere length (TL) in individuals with DSD? Summary answer Low autophagy activity and androgen receptor signaling stabilize GAR1, a telomerase-specific protein, leading to an increase in TL in DSD cells. What is known already Increased genome instability is linked to infertility, and compromised telomere maintenance is associated with genome instability. Individuals with DSD, characterized by dysgenic gonads and a high risk of Germ Cell Tumor (GCT) development, exhibit concurrent genome instability, increased TL, and deregulated Androgen Receptor (AR) signaling. Telomeric DNA damage promotes autophagy, which induces cell death and prevents propagation of replication crisis and tumorigenesis. We previously demonstrated that inhibition of autophagy, a conservative process of protein degradation, led to TL increase in cell lines derived from individuals with DSD, but the underlying mechanisms remain unclear. Study design, size, duration 112 individuals with DSD, including Swyer syndrome (46,XY females), Complete Androgen Insensitivity Syndrome (CAIS 46,XY females), Turner (45,X0 females), and Mixed Gonadal Dysgenesis (MGD 45,X0/46,XY females and males) were enrolled into study during routine praxis in our clinic. The samples were collected with written patients consent. A control group included fertile women and men, when appropriate, of comparable age. Samples underwent standard genetic and histological characterization. Lymphoblastoid cell lines were used for functional studies. Participants/materials, setting, methods Study groups comprised individuals with CAIS (n = 19); Swyer (n = 8); MGD (n = 12); Turner (n = 23) with mosaic (n = 12), with chr. X/Y deletions (n = 8), with X-isochr./idiocentric/marker chr. (n = 23) syndromes. Blood samples were collected for DNA, RNA and protein isolation from leukocytes. Additionally, individuals with DSD who underwent gonadectomy formed an additional group with histologically proven GCT (DSD-GCT, n = 7). Gonadal tissue was provided by the tissue bank. Samples were analyzed using quantitative real-time PCR (qRT-PCR), immunoblotting, and mass spectrometry. Main results and the role of chance Leukocytes from Swyer (p = 0,0069), Turner (p = 0,0064), and MGD without GCT (p = 0,05) groups exhibited a 1,5-folds increase in TL, diminished with GCT (p = 0,003). The RNA component of telomerase, TERC, was inhibited two times in Swyer (p = 0,0417). Proteomic analysis identified a telomerase protein GAR1 accumulation in Swyer, but downregulated in GCT group together with AR upregulation (p = 0,0379). Immunoblotting confirmed a two-fold increase of GAR1 in Swyer (p = 0,0022) and Turner (p = 0,0401), while GAR1 decreased in all DSD samples (p = 0,0455). GAR1 protein increased stability, not due to upregulated transcription, and promoted TL since we illustrated GAR1 accumulation within 72h of BafilomycinA1 treatment inhibiting autophagy. This treatment induced telomerase activity within 24h. Further autophagy inhibition caused accumulation of DNA damage in DSD cells, measured by γH2AX levels labeling double strand brakes (p = 0,0286). Dysgenic gonadal tissue analysis also showed a negative correlation between autophagy and TL-specific gene expression, with LC3 (p = 0,0341) and P62 (p = 0,0342) increased in all DSD samples, and proteins LC3 (p = 0,0298) and P62 (p = 0,0237) upregulated, and GAR1 (p = 0,0432) inhibited exclusively in Swyer group. Inhibition of AR increased TL (p = 0,0357) together with GAR1 (p = 0,0125) in DSD group at 19h, which was associated with a consecutive γH2AX upregulation (p = 0,0489) at 24h with Enzalutamide. Limitations, reasons for caution This study is based on a limited number of samples from the individuals with rare genetic syndromes, some observed only in 1 out of 10,000 clinical cases. Therefore, in vitro study models are recommended for further research. Wider implications of the findings The study reveals gene expression patterns and molecular regulatory mechanisms associated with DSD and GCT risk. The data suggest possibilities for prophylactic treatment of DSD individuals and new diagnostic approaches of GCT using peripheral blood. The study also emphasizes the importance of addressing genome integrity in infertility research. Trial registration number NA

Primary Co-Occurrence of Gonadal and Extragonadal Central Nervous System (CNS) Germ Cell Tumors (GCTs): Case Report and Review of the Literature

Background: The primary co-occurrence of gonadal and extragonadal central nervous system (CNS) germ cell tumors (GCTs) has rarely been reported in the literature, and a common opinion on the underlying etiopathogenetic mechanism is lacking. Objective: We aim to investigate the pathophysiological mechanisms and genetic pathways underlying the primary co-occurrence of gonadal and CNS GCTs. Methods: We reviewed data from 29 consecutive patients with a diagnosis of CNS GCT, evaluated in our Hospital over the past 23 years, and searched for those who had at least a primary gonadal co-occurrence of GCT. A systematic review of the literature according to the PRISMA guidelines was also conducted. For a comprehensive and detailed search, PubMed, Ovid MEDLINE, and Ovid EMBASE databases have been consulted. Boolean operators and MeSH terms were used to find studies. Only articles published between 2000 and 2023 were considered. Results: Including our Institutional case report, a total of 7 patients with both testicular NGGCTs and CNS GCTs were identified (5 patients with metachronous tumors and patients with synchronous presentation). The average age at tumor diagnosis was 17 years. The cerebral histotypes reported were mixed GCTs (3 cases; 43%), pure germinomas (3 cases, 43%), and one yolk sac tumor (14%). Two out of seven cases (29%) were syndromic, one suffering from Down Syndrome and the other from Testicular Dysgenesis Syndrome. Regarding the etiology and molecular mechanism of GCT development, several gene mutations have been reported in the literature. Particularly, genetic alterations in the MAPK and/or PI3K/AKT/mTOR pathway, together with mutations of the KIT gene, have been shown to guarantee survival and transformation of mismigrated totipotent primordial germ cells, while suppressor genes allow their resistance against apoptotic death. Aberrant chromosomes have also been reported to be responsible for oncogenic transformation. It is also known that CNS and testicular GCTs share some genetic/epigenetic profiles. Conclusions: The primary co-occurrence of testicular NGGCT and extragonadal CNS GCTs is extremely rare. Genetic factors seem to play a paramount role in their etiopathogenesis. Additional research is needed to elucidate molecular mechanisms of oncogenesis in such patients.

Effect of 900 MHz radiofrequency electromagnetic radiation emitted from mobile phone on testicular immunity and the associated risk of testicular germ cell tumor

The emission of radiofrequency electromagnetic radiation (RF-EMR) from mobile phones has been implicated in causing inflammatory changes in the testis. Nevertheless, the direct association of these changes with the development of testicular germ cell tumors (TGCT) remains unclear. Therefore, we purposed to investigate the effect of RF-EMR exposure on inflammatory changes in the testis, cytokine gene expression levels, and the incidence of TGCT. Twenty male Wistar albino rats were randomly assigned to either the study or control groups. The study group was exposed to RF-EMR at 900 MHz, 26 V/m, and a specific absorption rate (SAR) of 0.14 W/kg for 4 h/day over 8 weeks. Histopathological analysis, vitality analysis using Annexin V, and real-time polymerase chain reaction for the analysis of interleukin 1 (IL-1), IL-4, IL-10, tumor necrosis factor-alpha, and interferon-gamma (IFN-γ) cytokine gene expressions were performed on the testicular tissue. The median testis weight (163.0 g [133.0 – 183.0] vs. 179.0 g [134.0 – 195.0], P = 0.012) and volume (0.95 cm3 [0.800 – 1.400] vs. 1.100 cm3 [1.050 – 1.500], P = 0.031) of the study group were significantly lower compared to the control group. The seminiferous tubule damage (P < 0.001) and interstitial edema (P = 0.042) were significantly higher in the study group. The tunica albuginea thickness was significantly reduced in the study group (P < 0.001). The fold changes in the expression levels of IL-4 and IFN-γ increased significantly in the study group. Our findings indicate that RF-EMR exposure causes structural, histopathological, and inflammatory toxic effects on the testis. The observed elevation in gene expression levels of IL-4 and IFN-γ cytokines following RF-EMR exposure suggests their potential role as regulators of TGCT initiation, thereby offering a viable potential therapeutic target in combination with current treatments. Nonetheless, future well-designed studies are necessary to validate our findings.

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).

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.

Immune checkpoint inhibitors and Chimeric Antigen Receptor (CAR)-T cell therapy: Potential treatment options against Testicular Germ Cell Tumors.

Germ cell tumors (GCTs) represent a heterogeneous neoplasm family affecting gonads and rarely occurring in extragonadal areas. Most of patients have a good prognosis, often even in the presence of metastatic disease; however, in almost 15% of cases, tumor relapse and platinum resistance are the main challenges. Thus, novel treatment strategies with both improved antineoplastic activity and minor treatment-related adverse events compared with platinum are really expected. In this context, the development and the high activity demonstrated by immune checkpoint inhibitors in solid tumors and, subsequently, the interesting results obtained from the use of chimeric antigen receptor (CAR-) T cell therapy in hematological tumors, have stimulated research in this direction also in GCTs. In this article, we will analyze the molecular mechanisms underlying the immune action in the development of GCTs, and we will report the data from the studies that tested the new immunotherapeutic approaches in these neoplasms.

Prevalence, distribution, and risk markers for the development of gonadal germ cell tumors in patients with certain types of disorders of sexual differentiation with Y chromosome - A retrospective study.

To study the prevalence, subtypes, and risk markers for the development of gonadal germ cell tumors (GCT's) among disorders of sexual differentiation (DSD) patients with the Y chromosome. Design: A retrospective review of the patient's case records from 2010 to 2020 in Government Medical College, Thiruvananthapuram, India was studied. The study participants included 54 subjects with DSD containing the Y chromosome. Demographic data, external masculinization scoring, associated congenital anomalies, karyotyping, intraoperative findings such as gonadal location and internal genital ducts, histopathology of the resected gonads, and its immunohistochemistry were collected. The prevalence of gonadal GCT's was estimated from paraffin-embedded gonadectomy samples (S = 82). The median age of occurrence of gonadal GCT's was 18 years. The prevalence of malignant gonadal GCT's was highest among the PAIS group (19.2%) followed by gonadal dysgenesis (15.8% each in MGD and CGD) and least among CAIS (7.7%) (p < 0.01). The most common type of malignant gonadal GCT's in the descending order of frequency was dysgerminoma, seminoma, mixed GCT, and yolk sac tumor. Multivariance logistic analysis showed post-puberty and the presence of congenital anomalies were associated with the occurrence of gonadal GCT's ( P < 0.01). The overall prevalence of gonadal GCT's (malignant and premalignant) among DSD with Y chromosomes is nearly 25%. Dysgerminoma is the most common malignant gonadal GCT's. Age at or above 18 years and the presence of congenital anomalies like renal agenesis, retroperitoneal vascular defects, and congenital diaphragmatic hernia were independent risk markers for the development of gonadal GCT's.

OR05-1 Testicular Magnetic Resonance Imaging (MRI) Findings and Predictive Factors of Gonadal Neoplasia in Complete Androgen Insensitivity Syndrome

Abstract Introduction Complete Androgen Insensitivity Syndrome (CAIS) is characterized by a complete external genitalia appearance and testicular development in 46,XY individuals carrying inactivating mutations in the AR gene. Prophylactic gonadectomy versus saving gonads has been debatable in CAIS management, mainly due to the absence of an accurate biomarker for testicular germ cell tumors (GCT). Objective to cross the preoperative pelvic MRI data with the histological gonadal findings from CAIS patients who underwent prophylactic gonadectomy to identify predictive factors of GCT development. Methods We evaluated 30 gonads from 15 CAIS patients with proven AR mutation. All performed pelvic MRI before gonadectomy through the same MRI machine and were included in the MRI data analysis. Three blinded radiologists specialized in urology performed image analysis. The immunohistochemical markers PLAP, c-KIT, OCT3/4, SALL4, ER, alpha-inhibin, CD99, and calretinin were performed. Results Nine (60%) performed gonadectomy after puberty (&amp;gt;16 years old). We identified one case of classical seminoma and one case of Sertoli cell neoplasia (at 18 and 19 years of age, respectively). Overall, the prevalence of neoplasia was 13.3%. No case of premalignant intratubular germ cell neoplasia was detected.Histological data revealed a high prevalence of benign lesions (n=12; 80%). Leydig Cell Hyperplasia (67%), stromal fibrosis (53%), Sertoli Cell nodules (47%), hamartomatoums nodules (40%) and paratesticular cysts (67%) which are more frequent after puberty (p&amp;lt;.01). MRI detected heterogeneity in all cases harboring hamartomatoums nodules, Sertoli cell nodules, and testicular neoplasia. Benign lesions were bilateral in all cases. Regarding the MRI data, the presence of testicular cystic mass (by tumoral necrosis) on MRI and tumor size (higher among malignancies) correlated with malignancy (p=.008 and p=.03, respectively), whereas gonadal location (inguinal/abdominal), presence of solid nodules, T2WI and postcontrast features, nodule diffusion restriction, and presence of perigonadal cysts did not. Chronological age &amp;gt;16 years was associated with malignancy with a 4.3 OR (1.6–11.69). Conclusion Pelvic MRI is sensitive to detect gonadal nodules in CAIS patients, regardless of the gonad position, but most nodules are benign. Testicular malignancy should be suspicious in the presence of testicular cystic mass and large testis size in CAIS patients &amp;gt;16 years old. Presentation: Saturday, June 11, 2022 11:30 a.m. - 11:45 a.m.

Current view on testicular tumors from a developmental biological perspective : Important biomarkers and molecular pathological investigations

Germ cell tumors (GCTs) are the most common type of cancer in Germany in young men between 15 and 44years of age. The routinely performed diagnostic procedures are essential for the patient's treatment, but can be difficult due to heterogenous histologies. Additionally, the molecular mechanisms of the development of the special forms growing teratoma syndrome (GTS) and testicular tumors with malignant somatic transformation (MST) as well as of therapy resistance are not fully understood. Updated understanding of the molecular processes underlying GCT development and their special forms as well as recommendations for new and useful biomarkers. The development of GCTs is adynamic process largely influenced by the microenvironment. Seminomas (SEs) in particular seem to posses ahigher cellular plasticity than previously assumed, allowing SEs to be reprogrammed into an embryonal carcinoma (EC) or differentiate into extra-embryonal tissues (yolk sac tumors [YSTs], trophoblastic differentiation). Novel serological (mi371a-3p) and pathological (FOXA2) biomarkers are well suited to early detect GCTs and YSTs, respectively. For more aggressive tumors and special cases (GTS, MST), there are still no reliable diagnostics or specific/tailored therapies available. The ability of SEs to transit into EC or YSTs should be considered during therapy. Future research should focus on deciphering the special forms GTS and MST as well as the early recognition of YSTs, since their development seems to be an escape mechanism to chemotherapy.

Abstract 769: Genomic and transcriptomic characterization of benign and malignant struma ovarii

Abstract Background: Struma Ovarii (SO) is a rare ovarian teratoma characterized by the presence of thyroid tissue in &amp;gt;50% of the tumor. The majority of SO are benign; however, malignant transformation occurs in up to 5% of the cases. The molecular foundations of benign and malignant SO are grossly unknown. Therefore, the goal of this study was to perform the first comprehensive genomic and transcriptomic analysis of the benign and malignant SO. Material and Method: We performed whole-exome sequencing (WES) and targeted RNA-sequencing (seq) on the DNA and RNA extracted from formalin-fixed paraffin-embedded SO tumor tissue samples. WES library was prepared using Agilent’s SureSelect XT HS2 kit, with 4 samples failing the quality assessment (QA). Variants were called from GATK processed WES data and annotated using VEP (with ClinVar and COSMIC databases). The targeted RNA-Seq library was prepared using the TruSight RNA Pan-Cancer Panel kit covering 1385 cancer genes, with all samples passing QA. The clinical characteristics of the study cohort were summarized by percentages for categorical variables and medians with 25-75% interquartile ranges for continuous variables. Results: The study included 31 tissue samples - 21 benign and 10 malignant, including 6 cases of papillary thyroid cancer (PTC), 3 of follicular variant of PTC, and 1 of follicular thyroid cancer. Patients with benign SO were characterized by the median age at diagnosis of 39 years [33-54], tumor size of 3.1 cm [2.5-5.8], while the patients with malignant SO presented at age of 45 [28-54], tumor size of 6 cm [0.85-14] and metastatic disease in 30% (3/10) - 2 patients with peritoneal metastases and 1 patient with pelvic lymph node metastases. The E1A Binding Protein P300, EP300 (6/27), and Isocitrate dehydrogenase, IDH2 (5/27) were the topmost mutated genes in the SO samples. Malignant SO samples were characterized with the presence of pathogenic variants of KRAS (pQ61L and pG12V), NRAS (pQ61R), TP53 (splice site) mutations, and Nuclear Receptor Binding SET Domain Protein 1 (NSD1) fusion as the most common molecular drivers. Among benign SO samples, the most common driver was Thyroglobulin (TG) fusion with either Guanine Nucleotide binding protein (GNAS) or Rac Family Small GTPase 1 (RAC1). Differential expression analysis showed that the member of tumor suppressor family - tumor protein 63 (TP63) was the most downregulated (Log2FC = -3), while Double-sex and Mab-3 Related Transcription Factor 1 (DMRT1), implicated in the development of germ cell tumors, was the most upregulated gene in malignant SO samples over benign (Log2FC = 2.1; padj&amp;lt;0.05). Conclusions: In contrast to cancer arising from the thyroid gland, characterized by BRAFV600E as the most common mutation, malignant SO belongs to RAS-like tumors. The downregulation of tumor suppressors and upregulation of DMRT1 might be implicated in the malignant transformation of SO. Citation Format: Shilpa Thakur, Darryl Nousome, Kshama Aswath, Stephanie Cardenas, Sonam Kumari, Ruth Adewale, Maria Merino, Esra Dikoglu, Padmasree Veeraraghavan, Sriram Gubbi, Joanna Klubo-Gwiezdzinska. Genomic and transcriptomic characterization of benign and malignant struma ovarii [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 769.

Defining germline genetics of germ cell tumor: Implications for genetic testing and clinical management.

5007 Background: Epidemiologic studies on germ cell tumors (GCT) in men have shown GCT has a high heritability. Yet, the genetic mechanisms underlying development of GCT in men remain unknown. We sought to determine the prevalence of pathogenic or likely pathogenic (P/LP) germline variants in cancer predisposition genes in men with GCT and identify clinical and pathologic factors associated with the presence of P/LP variants. Methods: We retrospectively identified men with testicular (T) or mediastinal (M) GCT who underwent matched tumor--germline sequencing of ≥ 310 genes as part of an institutional genomic profiling initiative from 04/2015 to 12/2020. Presence of germline variants in clinically actionable genes associated with hereditary cancer syndromes were analyzed. Clinicopathologic characteristics, including primary site, histology and family history were assessed by the presence of P/LP variants and compared using Fisher’s exact test. Results: The study included 480 men, 70 (15%) with M-GCT and 410 (85%) with T-GCT. The median age of diagnosis was 30 years (interquartile range 24 – 39). A total of 81% had non-seminoma; 5% had localized disease; 46%, 14%, and 34% had good, intermediate, and poor risk advanced disease, respectively; 6% had family history of GCT. Among men with T-GCT, 4% had bilateral T-GCT. P/LP variants in clinically actionable genes were identified in 58 (12%) men in the entire cohort. Men with M-GCT had higher frequency of P/LP variants compared to those with T-GCT (15/70 [21%] vs 43/410 [10%], p = 0.02). A total of 7 (10%) men with M-GCT and 20 (5%) with T-GCT had P/LP variants in moderate or high penetrance cancer predisposition genes (p = 0.09). Most P/LP variants in both M-GCT (11/15, 73%) and T-GCT (33/43, 77%) were in genes involved in DNA damage repair pathways. The spectrum of moderate or high penetrance DNA damage repair genes identified is shown in the Table 1. Family history and histology (seminoma vs. non-seminoma) were not associated with the presence of a P/LP variants in the overall cohort (p = 0.8 and 0.7, respectively). Bilateral disease was not associated with the presence of P/LP variants in men with T-GCT (p = 0.4). Conclusions: Approximately 1 in 5 men with M-GCT carried a P/LP variant in a clinically actionable cancer susceptibility gene and 10% had a P/LP variant in a high-risk gene such as TP53 and PMS2. As men with GCT tend to develop disease in early adulthood, identification of germline P/LP variant has significant implications for enhanced cancer screening and surveillance, cascade testing for at-risk family members, and potential preconception genetic counseling. [Table: see text]

Identifying germline genetic alterations or environmental factors associated with bilateral germ cell tumor (GCT).

5035 Background: Germline genomic alterations and/or environmental factors are proposed as critical to GCT development given the significant rates of bilateral and familial cases. A number of low-moderate risk loci for GCT have been identified (Litchfield, Eur Urol 2018 &amp; Pluta, Nat Comm 2021), predominantly by analysing unilateral GCT, with a polygenic heritability model proffered. We postulate bilateral GCT represents an extreme phenotype with higher frequency of loss of function germline genomic alterations and/or pre-disposing environmental exposures. Methods: Bilateral GCT pts who consented to a prospective clinically annotated GCT DNA registry were included for analysis. The registry integrates self-reported questionnaires on environmental exposures, relevant medical history and germline whole exome sequencing (WES). Pathogenicity of variants identified by WES were classified using the algorithm Pathogenicity of Mutation Analyzer (PathoMAN) [Ravichandran, Genet Med 2019]. Aggregated WES results were curated to identify disruptive or deleterious variants affecting the same genes as the most significant individual variants in the Litchfield series. A &lt; 1% minor allele frequency threshold was set for candidate variants, likely benign/benign variants were excluded . Results: Of 43 bilateral cases with germline WES results, median age at first diagnosis was 32.1 years (range 18-73), 9 tumors were synchronous and 34 metachronous with median interval between diagnosis of 76.4 months for metachronous cases. Tumor histology was seminoma in 52 and nonseminoma in 34. 39 of 43 (91%) pts completed questionnaires with 35 (90%) reporting White race, 2 Hispanic/Latino, 1 Asian, and 1 Peruvian. 13 (33%) reported prior smoking, 21 (54%) prior marijuana use, and 6 (15%) other illicit drug use. 6 (15%) pts reported undescended testis and 1 (3%) hypospadias. 24/43 (45%) patients harbored ≥1 variant within 15 Litchfield genes (60%). 43 distinct variants were identified: 33 (77%) nonsynonymous missense alterations, 7 (16%) intron variants, 1 inframe deletion in STH3TC1, and 1 initiator and 1 stop codon variant in DEFB132, which plays a role in binding spermatozoa. Multiple bilateral patients harbored variants in MPDZ (n = 7), EHBP1L1 (n = 6), SKIV2L (n = 5), FAM160A2 (n = 3), ADAMTS18 (n = 3), JMJD4 (n = 3), ABCC4 (n = 3), R3HCC1 (n = 2), MLXIP (n = 2) and VPS16 (n = 2). Conclusions: In this bilateral GCT cohort, we identified several alterations in candidate susceptibility genes from the Litchfield series, whereas no convincing causative recurrent environmental exposures were observed. Our results support a polygenic model of GCT pathogenesis and show that analyzing enriched cohorts such as bilateral GCT may aid understanding of GCT pathogenesis. Additional analysis of the specific variants is required to further assess pathogenicity, elucidate their role and association with bilateral GCT.

Sex chromosome DSD individuals with mosaic 45,X0 and aberrant Y chromosomes in 46,XY cells: distinct gender phenotypes and germ cell tumour risks§

“Differences of Sexual Development (DSD),” individuals with rearranged Y chromosome breaks in their 46,XY cells are reported with male and female gender phenotypes and differences in germ cell tumour (GCT) risk. This raised the question of whether male or female gender and GCT risk depends on the site of the break and/or rearrangement of the individual´s Y chromosome. In this paper, we report molecular mapping of the breakpoint on the aberrant Y chromosome of 22 DSD individuals with a 45,X/46,XY karyotype reared with a different gender. Their Y chromosome breaks are found at different sites on the long and short Y arms. Our data indicate that gender rearing is, neither dependent on the site of Y breakage, nor on the amount of 45,X0 cells in the individuals' leukocytes. Most prominent are secondary rearrangements of the Y chromosome breaks forming di-centric Y-structures (“dic-Y”). Duplications of the short Y arm and the proximal part of the long Y arm are the results. A putative GCT risk has been analysed with immunohistochemical experiments on some dysgenetic gonadal tissue sections. With specific antibodies for OCT3/4 expression, we marked the pluripotent germ cell fraction being potential tumour precursor cells. With specific antibodies for DDX3Y, TSPY, and UTY we analyzed their putative Gonadoblastoma Y (GBY) tumour susceptibility function in the same specimen. We conclude GBY expression is only diagnostic for GCT development in the aberrant germ cells of these DSD individuals when strong OCT3/4 expression has marked their pluripotency.

The Polymorphisms of Genes Encoding Catalytic Antioxidant Proteins Modulate the Susceptibility and Progression of Testicular Germ Cell Tumor.

Simple SummaryTesticular cancer is the most common malignancy in the population of young and reproductively active men. The risk factors for its occurrence are not fully elucidated. Undescended testicle remains the main risk factor; however, more precise molecular studies associate genetic variations with susceptibility to testicular tumor development and progression. In this study, we found that specific variations in genes encoding antioxidant defense proteins confer risks of testicular cancer development and progression and, therefore, helps to identify subjects at higher risk, as well as those requiring additional diagnostics and more intensive forms of treatment.The simultaneous analysis of redox biomarkers and polymorphisms encoding for regulatory and catalytic antioxidant proteins was performed in order to evaluate their potential role in the development of testicular germ cell tumor (GCT), as well as the progression of the disease. NRF2 (rs6721961), GSTM3 (rs1332018), SOD2 (rs4880) and GPX3 (rs8177412) polymorphisms were assessed in 88 patients with testicular GCT (52 with seminoma) and 88 age-matched controls. The plasma levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), thiol groups and the plasma activity of glutathione peroxidase were measured. A significant association between variant GPX3*TC+CC genotype and risk of overall testicular GCT, as well as seminoma development, was found. Moreover, carriers of variant SOD2*TT genotype were at almost 3-fold increased risk of seminoma development. Interestingly, combined SOD2*TT/GPX3*TC+CC genotype conferred a 7-fold higher risk for testicular GCT development. Finally, variant GSTM3*AC+CC genotype was associated with a higher risk for the development of advanced diseased. The presence of assessed genetic variants was not associated with significantly higher levels of redox biomarkers in both testicular GCT patients, as well as in those diagnosed with seminoma. In conclusion, the polymorphic expression of certain antioxidant enzymes might affect susceptibility toward testicular GCT development, as well as the progression of the disease.

A novel case of prolonged Ifosfamide encephalopathy and long-term treatment with methylene blue: a case report and review of literature

BackgroundEncephalopathy following Ifosfamide treatment is a well-described phenomenon that is typically treated with Methylene Blue (MB). Chloroacetaldehyde, a potentially neurotoxic metabolite of Ifosfamide is hypothesized to cause this encephalopathy. Current guidelines for treatment is to stop Ifosfamide and provide supportive care. MB acts to inhibit Chloroacetaldehyde formation and has been described as a therapy and prophylaxis for Ifosfamide-encephalopathy. MB is effective within 30 min and lasts up to 3 days. Prolonged encephalopathy and MB therapy has not been described in the literature as lasting longer than 30 days following treatment.Case presentationWe present the case of an 11-year-old female with autistic spectrum disorder and recurrent episodes of severe somnolence for 7 months following Ifosfamide therapy for her Non-Germinomatous Germ Cell Tumor (GCT). Periods of somnolence occurred prior to receiving cranial RT. Administration of MB gave immediate but limited response, with resolution of somnolence lasting 1-2 days between administrations. The somnolence could not be explained by neuroimaging or laboratory evaluation, but EEG indicated persistent encephalopathy.ConclusionA literature review determines that neurotoxicity is a side effect of Ifosfamide, but this effect has not been described persisting longer than 30 days. Our case continued to require treatment with MB for 7 months following cessation of therapy. We report these novel clinical findings, and hypothesize that there could be a genetic/metabolic component linking this reaction to Ifosfamide with the case patient’s pre-existing autism. This possible association may also correlate to the already-established link between autism and the development of GCTs. This hypothesis leads to further discussion on the suitable usage of Ifosfamide in children with co-morbidities and the necessity of screening prior to its usage.

An Update on the Role of PI3K/ PTEN/ AKT Signaling Pathways in the Generation of Germ Cell Along with their Implications in Germ Cell Tumors Besides Ovarian Impairment: A Narrative Review

Numerous studies have pointed that the PI3K/ PTEN/ AKT signaling pathways are the key controllers of generation of germ cells precursors labeled as Primordial germ cells in addition to sustenance of follicular pool. Earlier we have already highlighted the role of MAPK/ERK along with Hippo/MST signaling for escalating of effectiveness as well as avoidance of chemo resistance in various Cancer Therapies besides modes by which chemotherapy with utilization of alkylating Agents result in follicular activation if there was any further deletion of primordial follicles? along with use of manipulation of the signaling pathways inclusive of PI3K/Akt pathway along with Hippo signaling path way in the Yes-associated protein (YAP) transcriptional coactivator with post synaptic density protein, drosophila disc large tumor suppressor [PDZ] and zonula occludens-1-binding motif (TAZ) -besides other controlling constituents in the generation of the EFs correlated with development of Early/ Primordial Follicles for the escalated ART results in premature ovarian insufficiency (POI) &amp; DOR patients. Here we concentrated on further updating the knowledge regarding germ cell tumors production besides reemphasizing on the dynamics of the ovarian Primordial germ cells pools besides decontrolling of these pathways In the generation of germ cell tumors along with ovarian impairment inclusive of, premature ovarian insufficiency (POI), premature ovarian failure (POF) &amp; diminished ovarian reserve (DOR) patients. Thus here we conducted a review utilizing search engine pubmed, google scholar; web of science; embase; Cochrane review library utilizing the MeSH terms like germ cells origin; Primordial germ cells; PI3K/PTEN/AKT signaling pathways; Hippo/MST pathways; mammalian target of rapamycin complex (mTORC); eukaryotic translation initiation factor 4E binding protein 1( (4E-BP1) c; TAZ; YAP ; MST1/2. We searched from 1990’s till date in 2022 We found 150 articles of which we used 62 articles for this narrative review or updating the pathways implicated and detail these PI3K/ PTEN/AKT signaling pathways mainly from point of view of Primordial germ cells development besides germ cell tumors development.

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.

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

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