Seminomatous Cell Research Articles

Pure intertubular seminoma (PITS) of the testis: A multi-institutional cohort of a rare growth pattern of seminoma

Pure intertubular seminoma (PITS) of the testis is described as the presence of seminoma cells within the interstitium of testis without any evidence of diffuse growth pattern or mass lesion of classical seminoma. These tumors are clinically and grossly inconspicuous and are diagnosed incidentally or during investigations for testicular pain, infertility or other symptoms. Rarely metastasis is the first presentation. Microscopic identification can be difficult and poses a diagnostic challenge in the absence of a mass lesion. Seminomas with exclusive intertubular growth patterns were gathered in an international cohort. Diagnoses were confirmed by fellowship-trained or specialized urologic pathologists. Cases with the presence of a classical diffuse or nested pattern of seminoma or any other germ cell tumor component were excluded. The patient's age, tumor characteristics and additional clinicopathologic features were recorded and analyzed. 15 patients of pure intertubular seminoma (PITS) were collated. The mean age of presentation was 29 years. Patients presented with variable symptoms, including undescended testis (26%, n = 4/15), testicular heaviness/pain (20%, n = 3/15) infertility (20%, n = 3/15) and metastasis (6%, n = 1/15); presentation was unknown in 4 patients. Of note, none of the patients presented because of testicular mass. Serum markers were within normal limits in most patients (93%, n = 14/15) with available data. No tumors were identified macroscopically; however, an ill-defined, grey-white, firm area was noted in one orchiectomy specimen. Microscopically, tumor cells were seen in intertubular spaces as dispersed individual cells or small clusters. Tumor cells were round to polygonal with large nuclei and prominent nucleoli. Mild to moderate lymphocytic infiltrates were seen admixed with tumor cells in 40% (n = 6/15) of the tumors. GCNIS was present in association with most PITS (73%, n = 11/15). Tubular atrophy with thickening of the basement membrane and Leydig cell hyperplasia was observed in one tumor. Thirty-three percent (n = 5/15) of the tumors showed pagetoid involvement of rete testis, including the tumor with metastasis. All tumors showed the classical immunohistochemical profile of seminoma, with PLAP, c-KIT, OCT3/4, D2-40 and SALL4 positivity. PITS can be clinically & pathologically inconspicuous, difficult to stage and liable to be misdiagnosed especially if presented with metastasis. Despite the inconspicuousness, PITS may represent an aggressive growth pattern of seminoma with the propensity for rete testis invasion.

Survival outcomes and molecular drivers of testicular cancer in hispanic men

ObjectiveTo examine survival outcomes and molecular drivers in testis cancer among Hispanic men using a large national sample and molecular database. MethodsWe reviewed the SEER registry for testicular cancer from 2000 to 2020. Cox proportional hazards models were used to examine the relationship between race/ethnicity and cancer-specific survival (CSS) by tumor type (seminoma vs. nonseminomatous germ cell tumors [NSGCT]). All models were adjusted for demographic, socioeconomic, and treatment variables. We accessed somatic mutations for testicular cancers through AACR Project GENIE v13.1 and compared mutational frequencies by ethnicity. ResultsOur cohort consisted of 43,709 patients (23.3% Hispanic) with median follow-up 106 months (interquartile range: 45-172). Compared to Non-Hispanic Whites (NWH), Hispanics presented at a younger age but with more advanced disease. Hispanics experienced worse CSS for NSGCT (HR 1.7, 95% CI: 1.5-2.0, P < 0.01) but not seminoma. Somatic mutation data was available for 699 patients. KIT and KRAS mutations occurred in 24.2% and 16.9% of seminoma patients (n = 178), respectively. TP53 and KRAS mutations occurred in 12.1% and 7.9% of NSGCT patients (n = 521), respectively. No differences in mutational frequencies were observed between ethnic groups. There was significant heterogeneity in primary ancestral group for Hispanic patients with available data (n = 53); 14 (26.4%) patients had primary Native American ancestry and 30 (56.6%) had primary European ancestry. ConclusionsCancer-specific survival is worse for Hispanic men with non-seminoma of the testicle. Somatic mutation analysis suggests no differences by ethnicity, though genetic ancestry is heterogeneous among patients identifying as Hispanic.

Unraveling the testicular seminoma tumor microenvironment by single-cell RNA-sequencing.

522 Background: Seminomas is a malignant germ cell tumor (GCT) affecting male adults and is characterized by low somatic mutation burden, histological homogeneity, and high survival rate. It is currently unknown what drives tumorigenesis of this curable testicular cancer. Tumor/immune microenvironment (TME) has a vital role in cancer survival and progression with implications in cell death resistance and immune escape. Herein, we aimed to unravel the transcriptomic TME landscape of seminomas and better understand seminoma pathogenesis. Methods: We performed single cell RNA-sequencing (scRNA-seq; 10X-Illumina platform) on surgically resected pure seminomas from four patients. A total of ~24,000 single cells were analyzed using Seurat/Bioconductor R packages and standard means for data visualization. Key findings were validated by in situ hybridization RNA analysis (RNAscope) on histological sections from these samples. Results: T-cells and Natural-Killer cell populations were mostly predominantly enriched in this seminoma cohort, where tumor cells consisted of a distinct subpopulation with limited presence (1%). Other distinct cell populations were also identified for additional immune cell types e.g., B-cells, macrophages/monocytes, endothelial and smooth muscle cells. ScRNA-seq revealed a unique set of signature marker genes (e.g., POU5F1, KHDC3L, DPPA5, NANOS3) highly expressed in seminoma cells which are associated with pivotal cellular processes e.g., developmental pluripotency and germ cell development. Notably, this gene set was also highly differentially expressed (DE) in The Cancer Genome Atlas (TGCA) seminomas (n=73) compared to TCGA non-seminomas (n=38) (FDR&lt;0.01). Amongst top marker genes, KHDC3L, also higher expressed in seminomas compared to normal testis, was validated by RNA-scope. KHDC3L demonstrated the strongest positive correlation with DPPA5 (Spearman/Pearson; R2=0.8, p&lt;0.0001), top #3 ranked DE gene as well as with other top 20 DE genes identified by scRNA-seq. Remarkably, similar strong correlations among other top DE genes were also found in the testicular TGCA cohort. Top DE genes including master transcription factors (e.g., POU5F1, NANOG, SOX4) are involved in the same protein pathway/networks likely suggesting synergic effect/involvement in the same regulatory circuitry. Gene Set Enrichment (GSEA) analyses demonstrated enrichment in immune signatures and pivotal pathways e.g., Wnt/beta catenin signaling and MYC targets. Conclusions: Our analyses revealed novel marker genes and immune signatures, uniquely expressed in seminomas which consist of a homogeneous cell population across all 4 tumors analyzed, clearly distinct from other cell types identified.

SMARCB1-Deficient Skull Base Chondrosarcoma with 12p Duplication Presenting as Somatic-Type Malignancy Arising from Metastatic Seminoma

Somatic-type malignancy (STM) can occur infrequently within a primary or metastatic testicular germ cell tumor (TGCT) and is associated with dismal prognosis and survival. STM with chondrosarcomatous features is exceedingly rare and head and neck involvement has not been previously documented. A 39-year-old white man presented with nasal obstruction and epistaxis. Imaging disclosed a 6.9-cm expansile tumor involving the nasal cavity and skull base with intraorbital and intracranial extension. The histopathologic properties of the tumor were compatible with chondrosarcoma, grade II-III. Immunohistochemically, malignant cells were strongly and diffusely positive for S100 and epithelial markers, and showed loss of SMARCB1 expression. IDH1/2 mutations were not detected. Following whole-body PET scan, a 7.0-cm left testicular mass was discovered and diagnosed as seminoma with syncytiotrophoblastic cells, stage pT3NXM1b. Extensive retroperitoneal, mediastinal, and supraclavicular lymphadenopathy was also noticed. Histopathologic examination of the left supraclavicular lymph node revealed metastatic seminoma. By FISH, most metastatic nodal seminoma cells harbored 1 to 4 copies of isochromosome 12p, while the chondrosarcoma featured duplication of 12p. Presence of a malignant TGCT with disseminated supradiaphragmatic lymphadenopathy, the unique immunophenotypic properties of the skull-based chondrosarcoma and lack of IDH1/2 aberrations with gain of 12p strongly support the diagnosis of STM chondrosarcoma arising from metastatic TGCT. The patient did not respond to chemotherapy and succumbed three months after diagnosis. Although exceedingly uncommon, metastasis to the head and neck may occur in patients with TGCT. This case of STM chondrosarcoma demonstrated divergent immunophenotypic and molecular characteristics compared to “typical” examples of head and neck chondrosarcoma. High index of suspicion is advised regarding the diagnosis of lesions that present with otherwise typical histomorphology but unexpected immunohistochemical or molecular features.

Intramural and Adapted-Germ Cell Neoplasia In situ

Germ cell neoplasia in situ emerges as a precursor lesion configuring type II germ cell tumours as testicular seminoma or post-pubertal non-seminomatous testicular germ cell tumours. Lesion is comprised of neoplastic gonocyte-like cells, latent totipotent or naive germ cells with developmental potential situated within ‘spermatogonial niche’ of seminiferous tubules. Germ cell neoplasia in situ delineates an increased incidence with conditions as uncorrected cryptorchidism, ambiguous genitalia, infertility or preceding history of post-pubertal germ cell tumour within contralateral testis. Neoplasm demonstrates aneuploidy or polypoid genotype with additional chromosomal gains as is chromosome 12p upon commencement of neoplastic invasion. Neoplastic cells appear enlarged, atypical, gonocyte-like and are incorporated with abundant, clear cytoplasm, enlarged, hyper chromatic nuclei, coarse nuclear chromatin, angulated cellular margins and prominent nucleoli.

Single-cell multi-omics analysis of human testicular germ cell tumor reveals its molecular features and microenvironment

Seminoma is the most common malignant solid tumor in 14 to 44 year-old men. However, its molecular features and tumor microenvironment (TME) is largely unexplored. Here, we perform a series of studies via genomics profiling (single cell multi-omics and spatial transcriptomics) and functional examination using seminoma samples and a seminoma cell line. We identify key gene expression programs share between seminoma and primordial germ cells, and further characterize the functions of TFAP2C in promoting tumor invasion and migration. We also identify 15 immune cell subtypes in TME, and find that subtypes with exhaustion features were located closer to the tumor region through combined spatial transcriptome analysis. Furthermore, we identify key pathways and genes that may facilitate seminoma disseminating beyond the seminiferous tubules. These findings advance our knowledge of seminoma tumorigenesis and produce a multi-omics atlas of in situ human seminoma microenvironment, which could help discover potential therapy targets for seminoma.

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

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

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

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

Extragonadal Germ Cell Tumors: A Single Institution Experience with Clinicopathological Correlation.

Background. Extragonadal germ cell tumors (EGCTs) are a rare heterogeneous group of tumors without evidence of primary gonadal germ cell tumors. They account for 2%-5% of overall malignancies. EGCTs are often not clinically suspected, making them challenging for pathologists. In this retrospective observational study, we describe our institutional experience among EGCTs with clinicopathological correlation. Materials and methods. All patients diagnosed as EGCTs from January 2014 to April 2023 were collected. All relevant clinical data and serum markers were retrieved from hospital medical records. Histopathology and immunohistochemistry slides were reviewed. Results. The present study included a total of 56 patients; 34 (60%) men and 22 (40%) women with a men-to-women ratio of 1.5:1. Of them, 1 patient had congenital/neonatal EGCTs, 21 patients had prepubertal EGCTs, and 34 had post-pubertal EGCTs. The common sites included are mediastinum (45%), sacrococcyx (18%), retroperitoneum (14%), and central nervous system (12%). The other rare sites were the vagina, liver, colon, and duodenum. The common germ cell tumor included mature teratoma (34%), mixed germ cell tumor (27%), seminoma/germinoma (12%), pure yolk sac tumor (11%), immature teratoma (9%), mature teratoma with somatic tumor (5%), and embryonal carcinoma (2%). All histological diagnoses of germ cell tumors were confirmed with IHC markers like PLAP, CD117 (KIT), AFP, LIN28, CD30, and β-hCG. Pre and posttreatment serum tumor marker levels were available in 37 patients. All our treated patients had a decrease or normal tumor marker levels post-therapy. Conclusion. In our study, a heterogeneous group of germ cell tumors was seen. Most of them were seen in post-pubertal adolescents and young adults. Early intervention by platinum-based combination chemotherapy in seminoma and nonseminomatous germ cell tumors has significantly improved the prognosis of malignant EGCTs similar to their germ cell counterparts.

SAT481 Metastatic Choriocarcinoma, A Rare Cause of hCG-Mediated Hyperthyroidism

Abstract Disclosure: S. Jain: None. M. Crabtree: None. C. Houston: None. Background: Structural homology between human chorionic gonadotropin (hCG) and thyrotropin stimulating hormone (TSH) allows high levels of hCG to exert thyrotropic effects via TSH receptors. Although hCG-mediated hyperthyroidism is most frequently encountered in the context of pregnancy, it also occurs rarely as a paraneoplastic syndrome. We present a case of hCG-mediated hyperthyroidism due to metastatic choriocarcinoma in a postmenopausal woman. Case Presentation: A previously healthy 59-year-old woman was evaluated for shortness of breath and found to have multiple lung nodules concerning for metastatic malignancy. Further imaging demonstrated an enlarged heterogeneous uterus with an enlarged endometrial cavity. Endometrial biopsy revealed poorly differentiated carcinoma with some areas of possible squamous differentiation. Subspecialty cytopathology review was requested. One week after biopsy, the patient was brought to the emergency department with acute hypoxic respiratory failure following a seizure. She was found to be febrile, tachycardic, hypertensive, and minimally responsive. Head CT revealed a left frontal brain mass with surrounding vasogenic edema, concerning for metastasis. Laboratory evaluation revealed TSH of 0.03 mIU/L (normal 0.35-4.94) and free T4 of 2.30 ng/dL (normal 0.70-1.48). Thyroid stimulating immunoglobulin was negative. Beta-hCG was found to be significantly elevated at 982,093 mIU/mL after 1:100 dilution. She was started on treatment with propranolol, dexamethasone, and chemotherapy with etoposide and cisplatin. Meanwhile, cytopathology review identified scattered cells with features of choriocarcinoma and positive hCG immunostaining, felt to represent a diagnosis of poorly differentiated choriocarcinoma. After one cycle of chemotherapy, the patient’s beta-hCG decreased to 477,668 mIU/mL and free T4 decreased to 1.05 ng/dL. Due to progressive worsening of her clinical status, she was transitioned to palliative care and passed away on hospital day 14. Discussion: Hyperthyroidism mediated by hCG is most often seen in patients with hyperemesis gravidarum, gestational transient thyrotoxicosis, or gestational trophoblastic disease. Our case underscores the importance of considering hCG-mediated hyperthyroidism outside the context of pregnancy. Rare paraneoplastic sources of hCG include choriocarcinoma, seminoma, and other germ cell tumors. Hyperthyroidism in these cases is usually subclinical or mild, but instances of thyroid storm have been reported with metastatic disease. Beta blockers, glucocorticoids, and thionamides can be used as temporizing measures, but definitive therapy requires treatment of the causative malignancy. Presentation Date: Saturday, June 17, 2023

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

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

ESX1 gene as a potential candidate responsible for male infertility in nonobstructive azoospermia

Infertility is a problem that affects approximately 15% of couples, and male infertility is responsible for 40–50% of these cases. The cause of male infertility is still poorly diagnosed and treated. One of the prominent causes of male infertility is disturbed spermatogenesis, which can lead to nonobstructive azoospermia (NOA). Whole-genome sequencing (WGS) allows us to identify novel rare variants in potentially NOA-associated genes, among others, in the ESX1 gene. The aim of this study was to activate the ESX1 gene using CRISPRa technology in human germ cells (testicular seminoma cells—TCam-2). Successful activation of the ESX1 gene in TCam-2 cells using the CRISPRa system was achieved, and the expression level of the ESX1 gene was significantly higher in modified TCam-2 cells than in WT cells or the negative control with nontargeted gRNA (p < 0.01). Using RNA-seq, a network of over 50 genes potentially regulated by the ESX1 gene was determined. Finally, 6 genes, NANOG, CXCR4, RPS6KA5, CCND1, PDE1C, and LINC00662, participating in cell proliferation and differentiation were verified in azoospermic patients with and without a mutation in the ESX1 gene as well as in men with normal spermatogenesis, where inverse correlations in the expression levels of the observed genes were noted.

P-049 Verteporfin suppresses cell proliferation, survival and migration of TCam-2 Human Seminoma Cells via inhibits the YAP-TEAD complex

Abstract Study question Does verteporfin administration effect on proliferation, survival and migration of TCam-2 human seminoma cells? Summary answer Verteporfin may be a promising anticancer agent for TCam-2 human seminoma cells via inhibition of cell viability, proliferation, migration and stimulation of apoptosis. What is known already Seminoma is germ cell tumor of the testis and TCam-2 cells is the first seminoma-derived cell line that retains many of the characteristic traits of seminoma. The Hippo signalling is a evolutionarily highly conserved pathway that controls organ size, tissue homeostasis, and cancer development. In mammals, the Hippo pathway consists of the serine/threonine kinases Mammalian Sterile 20-protein Kinase 1 and 2 (MST1/2), Large Tumor Suppressor Homologues 1 and 2 (LATS1/2), Yes-Associated Protein (YAP), and its transcriptional cofactor TAZ. Verteporfin inhibits YAP–TEAD complex, blocking cell proliferation and survival. Study design, size, duration TCam-2 cells were treated with different concentrations of verteporfin (1, 5, 10, 20 and 40 µM) for 24, 48 and 72 hours (h). Participants/materials, setting, methods TCam-2 cells were treated with verteporfin at different doses and incubation times. Afterwards, protein expression of Hippo pathway proteins was determined by western blot, mRNA levels were analyzed by qRT-PCR and protein localizations were evaluated by immunoflourescence staining. Cell migration was determinated with wound-healing scratch assay; cell viability, early-late apoptosis and necrosis rates were evaluated by flow cytometry. Main results and the role of chance We detected that 40 μM verteporfin decreased LATS1/2 and p-LATS1/2 protein expression after 48h and 72h treatment (p &amp;lt; 0.05, **p&amp;lt;0.01). MST1/2 and p-MST1/2 was significantly decreased in groups treated with 20 and 40 µM verteporfin after 48 and 72 h of treatment (p &amp;lt; 0.05, ****p&amp;lt;0.0001). YAP expression decreased at 5, 20 and 40 μM verteporfin treatment after 48h and all verteporfin concentrations after 72h (**p&amp;lt;0.01, ***p&amp;lt;0.001, ****p&amp;lt;0.0001). p-YAP expression significantly decreased at all verteporfin concentrations after 48h and only at 40 μM concentrations after 72h (**p&amp;lt;0.01). TEAD4 expression decreased at all verteporfin concentrations after 48 and 72h (****p&amp;lt;0.0001). After 48 and 72h of verteporfin treatment, no significant difference was observed in the expression of the MST1, MST2, LATS1, LATS2 and YAP1 mRNA levels in TCam-2 cells, while TEAD4 mRNA expression level was significantly decreased at 20 and 40 µM compared to control (**p&amp;lt;0.01, ***p&amp;lt;0.001). We also showed that Hippo signaling pathway proteins localized in the cell nucleus especially YAP1 and TEAD4, were translocated to cytoplasm depending on the increasing dose of verteporfin in TCam-2 cells. Verteporfin decreased cell proliferation (****p&amp;lt;0.0001), migration and cell viability (****p&amp;lt;0.0001), as well as increased the early-late apoptosis (**p&amp;lt;0.01, ***p&amp;lt;0.001, ****p&amp;lt;0.0001) in TCam-2 human seminoma cells dose-dependent manner. Limitations, reasons for caution The TCam-2 cell line was gifted by Prof. Dr. Hubert Schorle (Department of Developmental Pathology and Institute of Molecular Diagnostic Pathology, Faculty of Medicine, Germany, Bonn) to our laboratory. There is no limitation in the scope of the study. Wider implications of the findings We determined for the first time the effect of verteporfin treatment on the protein expression and mRNA levels of Hippo signaling pathway in TCam-2 human seminoma cells. We suggested that Hippo pathway and verteporfin relationship may be a therapeutic target for seminoma by affecting cell proliferation, survival, migration and apoptosis. Trial registration number not applicable

Updating germ cell tumour pathogenesis - the ability of seminomas for FOXA2-driven extra-embryonic differentiation.

Testicular germ cell tumours are the most common solid malignancies in young men of age 14-44 years. It is generally accepted that both seminomas and non-seminomas arise from a common precursor, the germ cell neoplasia in-situ, which itself is the result of a defective (primordial) germ cell development. The stem cell-like population of the non-seminomas, the embryonal carcinoma, is capable of the differentiation of all three germ layers (teratomas) and extra-embryonic tissues (yolk-sac tumours, choriocarcioma) into cells. In contrast, seminomas are thought to have a limited differentiation potential. Nevertheless, several studies have highlighted their ability to undergo reprogramming to an embryonal carcinoma or differentiation into other non-seminomatous entities. Here, we demonstrate that in approximately 5% of seminomas, the yolk-sac tumour driver gene FOXA2 is detectable at the protein level, indicative of an occult yolk-sac tumour subpopulation that putatively arose from seminoma cells, as the presence of other GCT entities could be excluded. The presence of these subpopulations might render the tumour more aggressive and argue for an adjustment of the therapeutic concept. We used our data to update the model of germ cell tumour pathogenesis, especially regarding the developmental potential of seminomas. Additionally, we suggest to include detection of FOXA2 into standard routine diagnosis of seminomas.

TAMs PD-L1(+) in the reprogramming of germ cell tumors of the testis

BackgroundIn recent years, several studies focused on the process of reprogramming of seminoma (S) cells, which regulates the transition from pure S (P-S) to S component (S-C) of mixed germ cell tumors of the testis (GCTT) and finally to embryonal carcinoma (EC) and other nonseminomatous GCTT (NS-GCTT). The accepted pathogenetic model is driven and regulated by cells (macrophages, B- and T-lymphocytes) and molecules of the tumor microenvironment (TME). Herein, we tested a series of GCTT with double staining (DS) for CD68-PD-L1 to evaluate tumor-associated macrophages (TAMs) expressing programmed death-ligand 1 (PD-L1) [TAMs PD-L1(+)] and clarify if these cells may be involved in establishing the fate of GCTT. MethodsWe collected 45 GCTT (comprising a total of 62 different components of GCTT). TAMs PD-L1(+) were evaluated with three different scoring systems [TAMs PD-L1(+)/mm2, TAMs PD-L1(+)/mm2H-score, TAMs PD-L1(+) %], and compared using pertinent statistic tests (Student’s t-test and Mann-Whitney U test). ResultsWe found that TAMs PD-L1(+) values were higher in S rather than EC (p = 0.001, p = 0.015, p = 0.022) and NS-GCTT (p < 0.001). P-S showed statistically significant differences in TAMs PD-L1(+) values compared to S-C (p < 0.001, p = 0.006, p = 0.015), but there were no differences between S-C and EC (p = 0.107, p = 0.408, p = 0.800). Finally, we found statistically significant differences also in TAMs PD-L1(+) values between EC and other NS-GCTT (p < 0.001). ConclusionsTAMs PD-L1(+) levels gradually decrease during the reprogramming of S cells {P-S [(high values of TAMs PD-L1(+)] → S-C and EC [(intermediate values of TAMs PD-L1(+)] → other NS-GCTT [(low values of TAMs PD-L1(+)], supporting a complex pathogenetic model where the interactions between tumor cells and TME components [and specifically TAMs PD-L1(+)] play a key role in determining the fate of GCTT.

The Subtype Identity of Testicular Cancer Cells Determines Their Immunostimulatory Activity in a Coculture Model.

Testicular germ cell cancer (TGCC) is subdivided into several subtypes. While seminomatous germ cell tumors (SGCT) are characterized by an intensive infiltration of immune cells which constitute a pro-inflammatory tumor micromilieu (TME), immune cells in non-seminomatous germ cell tumors (NSGCT) are differently composed and less abundant. Previously, we have shown that the seminomatous cell line TCam-2 promotes T cell and monocyte activation in a coculture model, resulting in mutual interactions between both cell types. Here we set out to compare this feature of TCam-2 cells with the non-seminomatous cell line NTERA-2. Peripheral blood T cells or monocytes cocultured with NTERA-2 cells failed to secrete relevant amounts of pro-inflammatory cytokines, and significantly downregulated the expression of genes encoding activation markers and effector molecules. In contrast, immune cells cocultured with TCam-2 cells produced IL-2, IL-6 and TNFα, and strongly upregulated the expression of multiple pro-inflammatory genes. Furthermore, the expression of genes involved in proliferation, stemness and subtype specification remained unaltered in NTERA-2 cells during coculture with T cells or monocytes, indicating the absence of mutual interactions. Collectively, our findings uncover fundamental differences between SGCT and NSGCT in their capability to generate a pro-inflammatory TME, which possibly impacts the clinical features and prognosis of both TGCC subtypes.

Burned-out testicular germ cell tumors: a clinicopathological analysis of three cases

Objective: To investigate the clinicopathological features and possible mechanisms of burned-out testicular germ cell tumors. Methods: The clinical and imaging data, histology and immunophenotypic characteristics of three cases of burned-out testicular germ cell tumors diagnosed at the Ruijin Hospital, Medical College of the Shanghai Jiaotong University, from 2016 to 2020 were retrospectively analyzed. The relevant literature was reviewed. Results: The mean age of the three patients was 32 years. Case 1 had an elevated preoperative alpha-fetoprotein level (810.18 μg/L) and underwent "radical pancreaticoduodenectomy and retroperitoneal lesion resection" for a retroperitoneal mass. Postoperative pathology showed embryonal carcinoma, which needed to exclude gonadal metastasis. Color Doppler ultrasound showed a solid mass of the right testis, with hypoechoic lesion and scattered calcification in some areas. Case 2 was a "right supraclavicular lymph node biopsy specimen." Chest X-ray showed multiple metastases in both lungs. The biopsy showed metastatic embryonic carcinoma and bilateral testicular color Doppler ultrasound revealed abnormal calcifications in the right testicle. Case 3 showed a cystic mass of the right testis with calcification and solid areas. All 3 patients underwent radical right orchiectomy. Grossly, borders of the testicular scar areas were well defined. Cross sectioning of the tumors showed a gray-brown cut surface and single focus or multiple foci of the tumor. The tumor maximum diameter was 0.6-1.5 cm. Microscopically, lymphocytes, plasma cells infiltration, tubular hyalinization, clustered vascular hyperplasia and hemosiderin laden macrophages were found in the scar. Atrophic and sclerotic seminiferous tubules, proliferation of clustered Leydig cells and small or coarse granular calcifications in seminiferous tubules were present around the scar. Seminoma and germ cell neoplasia in situ were seen in case 1, germ cell neoplasia in situ was seen in case 2 and germ cells with atypical hyperplasia were seen in case 3. Immunohistochemistry showed that embryonic carcinoma expressed SALL4, CKpan(AE1/AE3) and CD30, seminoma and germ cell tumor in situ expressed OCT3/4, SALL4 and CD117, and spermatogenic cells with atypical hyperplasia expressed CD99 and SALL4. The Ki-67 positive index was about 20%, while OCT3/4 and CD117 were both negative. Conclusions: Burned-out testicular germ cell tumors are rare. The possibility of gonad testicular metastasis should be considered first for extragonadal germ cell tumor. If fibrous scar is found in testis, it must be determined whether it is a burned-out testicular germ cell tumor. The burned-out mechanisms may be related to the microenvironment of tumor immune-mediated and local ischemic injury.

Diagnosis and treatment of primary seminoma of the prostate: A case report and review of literature

Primary seminoma of the prostate (PSP) is a rare type of extragonadal germ cell tumour that is easily misdiagnosed, owing to the lack of specific clinical features. It is therefore necessary for clinicians to work toward improving the accuracy of PSP diagnosis. A 59-year-old male patient presenting with acute urinary retention was admitted to a local hospital. A misdiagnosis of benign prostatic hyperplasia led to an improper prostatectomy. Histopathology revealed PSP invading the bladder neck and bilateral seminal vesicles. Further radiotherapy treatment for the local lesion was performed, and the patient had a disease-free survival period of 96 mo. This case was analysed along with 13 other cases of PSP identified from the literature. Only four of the cases (28.6%) were initially confirmed by prostate biopsy. In these cases, imaging examinations showed an enlarged prostate (range 6-11 cm) involving the bladder neck (13/14). Of the 14 total cases, 11 (78.6%) presented typical pure seminoma cell features, staining strongly positive for placental alkaline phosphatase, CD117, and OCT4. The median age at diagnosis was 51 (range 27-59) years, and patients had a median progression-free survival time of 48 (range 6-156) mo after treatment by cisplatin-based chemotherapy combined with surgery or radiotherapy. The remaining three were cases of mixed embryonal tumours with focal seminoma, which had clinical features similar to those of pure PSP, in addition that they also had elevated serum alpha-fetoprotein, beta-human chorionic gonadotropin, and lactose dehydrogenase. PSP should be considered in patients younger than 60 years with an enlarged prostate invading the bladder neck. Further prostate biopsies may aid in proper PSP diagnosis. Cisplatin-based chemotherapy is still the main primary therapy for PSP.

MP33-17 EXAMINING SURVIVAL OUTCOMES BY RACE AND ETHNICITY IN TESTIS CANCER: A REVERSE HISPANIC PARADOX?

You have accessJournal of UrologyCME1 Apr 2023MP33-17 EXAMINING SURVIVAL OUTCOMES BY RACE AND ETHNICITY IN TESTIS CANCER: A REVERSE HISPANIC PARADOX? Michael Rezaee, Maximilian Pallauf, Pranjal Agrawal, Roy Elias, Sean Fletcher, Yasser Ged, and Nirmish Singla Michael RezaeeMichael Rezaee More articles by this author , Maximilian PallaufMaximilian Pallauf More articles by this author , Pranjal AgrawalPranjal Agrawal More articles by this author , Roy EliasRoy Elias More articles by this author , Sean FletcherSean Fletcher More articles by this author , Yasser GedYasser Ged More articles by this author , and Nirmish SinglaNirmish Singla More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003266.17AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The incidence of testis cancer among Hispanic men in the United States is increasing. Prior research has demonstrated that Hispanic patients exhibit a survival benefit for many medical conditions, a phenomenon known as the “Hispanic Paradox.” However, this paradox may not extend to testis cancer. We sought to examine survival outcomes in testis cancer by race using a large national sample. METHODS: A population-based retrospective cohort study of testis cancer patients was performed using Surveillance, Epidemiology, and End Results (SEER) data for the years 2000 through 2019. Cox proportional hazards models were used to examine the relationship between race/ethnicity and testis cancer-specific survival, both overall and by histologic type (seminoma vs. nonseminomatous germ cell tumors [NSGCT]). All models were adjusted for age, stage, receipt of postoperative radiation or chemotherapy, year of diagnosis, income, marital status, and rural status. RESULTS: The final analytic cohort consisted of 18,600 patients, of whom 66.8% were Non-Hispanic White (NHW), 2.2% Non-Hispanic Black (NHB), 24.9% Hispanic (all races), and 6.1% Non-Hispanic Asian or Pacific Islander (API). Median follow up was 92 months. Death from testis cancer occurred in 2.7% of patients. A greater proportion of Hispanics were diagnosed at an age less than 30 years (54.4%) and with initial metastatic disease (15.2%) compared to other groups. Hispanics experienced significantly worse cancer-specific survival (hazard ratio [HR] = 1.8, 95% CI: 1.5-2.2) compared to NHW patients overall (Figure 1). When stratified by tumor type, cancer-specific survival was significantly worse for Hispanics (HR = 1.8, 95% CI: 1.4-2.2) and API’s (HR = 1.6, 95% CI: 1.1-2.5) with NSGCTs. No difference was observed in cancer-specific survival by race/ethnicity for seminoma patients, however. CONCLUSIONS: Contrary to the Hispanic paradox reported across many medical conditions, cancer-specific survival is significantly worse for Hispanic men with testis cancer, particularly among those with NSGCT tumors. Future investigation of both public health and biological factors is needed to identify actionable drivers for this observed difference. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e457 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michael Rezaee More articles by this author Maximilian Pallauf More articles by this author Pranjal Agrawal More articles by this author Roy Elias More articles by this author Sean Fletcher More articles by this author Yasser Ged More articles by this author Nirmish Singla More articles by this author Expand All Advertisement PDF downloadLoading ...

Analysis of connexin 43, connexin 45 and N-cadherin in the human sertoli cell line FS1 and the human seminoma-like cell line TCam-2 in comparison with human testicular biopsies

BackgroundGerm cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression.MethodsFS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines’ representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling.ResultsCx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells.ConclusionThe junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression.