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Testicular germ cell tumors have the highest incidence in young men (between 15 and 44 years of age) and its etiology is still unclear, but its emergence on puberty suggests a hormone-dependent mechanism for the development of these tumors and their progression. We previously identified the estrogen receptor ESR1, ESR2, GPER and an isoform of ESR1, the ESR1-36 in human testicular embryonic carcinoma NT2/D1 cells, and the activation of SRC induced by ESR1 and ESR2 in these cells. Therefore, this study aimed to analyze the role of ER in the activation of ERK1/2, and the involvement of SRC and ERK1/2 on proliferation, migration, and invasion of the NT2/D1 cells. Our results showed that the activation of ESR1 (using ESR1-selective agonist PPT) or ESR2 (using ESR2-selective agonist DPN) increased phosphorylation of ERK1/2 in NT2/D1 cells. In the presence of the selective inhibitor for SRC-family kinases PP2, or the MEK specific inhibitor U0126, the effects of 17β-estradiol (E2) or PPT were blocked on proliferation and invasion of NT2/D1 cells. Finally, the proliferation, migration, and invasion of NT2/D1 cells simulated by E2 or ESR2 were also blocked by PP2 and U0126. This study provides novel insights into molecular mechanisms of ER in NT2/D1 cells by demonstrating that ER activates rapid responses molecules, including SRC and ERK1/2, which enhance the tumorigenic potential of testicular cancer cells., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2024. Published by Elsevier Inc.)

SummaryWe previously demonstrated that among various histological types of human testicular germinal cell tumors (GCTs), embryonal carcinoma (EC) preferentially expresses low-sulfated keratan sulfate (KS) consisting of repeating N -acetyllactosamine (LacNAc) disaccharide units composed of galactose and 6- O -sulfated N -acetylglucosamine (GlcNAc), which is recognized by the R-10G antibody. Recently, we generated another anti-low-sulfated KS monoclonal antibody, 294-1B1. Immunohistochemical analysis of testicular GCTs ( n =83) revealed that the low-sulfated KS recognized by 294-1B1 is also preferentially expressed in EC but minimally in other GCT histological types. Moreover, immunolabeling with R-10G and 294-1B1 antibodies was resistant to peptide- N -glycosidase F digestion, and EC was not stained with the MECA-79 antibody, indicating that low-sulfated KS expressed in EC contains mucin-type core 2 O -glycans carrying GlcNAc-6- O -sulfated oligo-LacNAc. Double immunofluorescence staining showed that R-10G and 294-1B1 antibody signals colocalized with those for podocalyxin (PODXL). Furthermore, western blot analysis of recombinant human PODXL•IgG fusion proteins secreted from low-sulfated KS-expressing human embryonic kidney 293T cells revealed that PODXL functions as a core protein for low-sulfated KS. Taken together, these findings strongly suggest that the PODXL glycoform decorated with low-sulfated KS is preferentially expressed in human testicular EC and may therefore serve as a diagnostic marker for this malignancy., Competing Interests: Competing InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Serpinb9 is an inhibitor of granzyme B and is potentially involved in the immune escape of tumor cells. In the present study, bioinformatics analysis using open databases suggested that SerpinB9 is overexpressed in testicular embryonal carcinoma. Immunohistological analysis was performed on 28 cases of testicular germ cell tumors to investigate the relationship between SerpinB9 expression in testicular germ cell tumors and the tumor immune environment. SerpinB9 was significantly upregulated in the non-seminoma group and inversely correlated with the number of tumor-infiltrating CD8-positive cells. In addition, yolk sac tumors were characterized by the loss of human leukocyte antigen-class I expression. These findings suggest that SerpinB9 contributes to the immune escape of testicular germ cell tumors. Targeting therapy for SerpinB9 might therefore be useful in immunotherapy for testicular germ cell tumors resistant to immune checkpoint inhibitors., (© 2023. The Author(s) under exclusive licence to The Japanese Society for Clinical Molecular Morphology.)

Background: Embryonal carcinoma is a rare tissue type in germ cell tumors. According to our literature review, metastatic embryonal carcinoma misdiagnosed as lymphoma because of its high similarity to lymphoma is extremely rare and has not been reported yet., Case Presentation: A 46-year-old middle adulthood male presented with unexplained fever, night sweats, abdominal distension for 3 months, and weight loss of around 7kg during almost 6 months, which is extremely similar to lymphoma from the clinical features and imaging examinations. After a clear diagnosis, the case not only obtained the opportunity of surgery but was also exempted from radiotherapy. The treatment effect was good. We report a case of rare metastatic embryonal carcinoma, which can provide insight into the diagnosis and treatment of embryonal carcinoma., Conclusion: Metastatic embryonal carcinoma of abdominal lymph nodes can be highly similar to lymphoma; the diagnosis can only be based on clinical manifestations and imaging examination but also combined with patient history, tumor markers and biochemical examination. However, the final diagnosis depends on pathological biopsy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Introduction: The purpose of this study was to perform a population-based investigation to assess the disease characteristics and prognosis of children and adolescents with malignant mediastinal germ cell tumors (MMGCT)., Methods: Data on the demographics, treatment, and survival outcomes of children and adolescents with MMGCT from January 1, 2000 to December 31, 2018 were obtained. To compare survival curves, the log-rank test was employed. The generation of survival curves based on different parameters was done using Kaplan-Meier estimations. Cox proportional hazards regression was performed to determine the variables linked to disease-specific survival., Results: The selection criteria were met by 152 MMGCT patients, 130 of whom were male. Fifty three cases of mixed germ cell tumors (GCTs), 41 cases of malignant teratomas, 26 cases of yolk sac tumors, 14 cases of seminoma, 13 cases of choriocarcinomas, and five cases of embryonal carcinoma were reported. Overall survival at 3 and 5 y for all patients was 63.1% and 61.2%, respectively. Malignant teratoma, yolk sac tumors, and mixed GCTs in children and adolescents had comparable survival rates, while those with choriocarcinoma and embryonal carcinoma showed the worst prognosis. Embryonal carcinoma, malignant teratoma, mixed GCTs, and choriocarcinoma were found as risk factors by multivariate Cox proportional hazards analysis. In contrast, surgery and younger age were protective factors. However, chemotherapy alone showed no survival benefits., Conclusions: Our population-based evidence showed that MMGCT had worse prognosis in older children and adolescents. Choriocarcinomas and embryonal carcinomas had the worst prognosis. Surgery can prolong survival time. Chemotherapy and radiotherapy were not associated with improved prognosis., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Introduction: Bilateral testicular tumors are very rare, accounting for 1%-5% of all testicular germ-cell tumors (TGCTs). The vast majority of primary bilateral TGCTs are metachronous, with synchronous tumors comprising approximately 0.5%-1% of all cases. Those occurring synchronously share mostly the same histological pattern, predominantly seminoma, with synchronous bilateral TGCTs (SBTGCTs) with discordant subtypes being extremely rare., Case Presentation: We present the case of a 20-year-old male complaining of a palpable painless right testicular mass incidentally noticed during sexual intercourse. Ultrasonography (US) and magnetic resonance imaging (MRI) of the scrotum demonstrated bilateral testicular lesions, while staging with contrast-enhanced computed tomography (CT) exhibited normal findings. Right radical orchiectomy and left testis-sparing surgery (TSS) with concomitant onco-testicular sperm extraction (onco-TESE) were initially performed. Histology of the right testis revealed a mixed germ-cell tumor, consisting of seminoma and embryonal carcinoma, while that from the left testis disclosed embryonal carcinoma and intratubular germ-cell neoplasia unclassified (IGCNU) infiltrating the surgical margins. Hence, left orchiectomy was subsequently scheduled with histology unveiling IGCNU in the greatest part of the remaining testicular parenchyma. Following adjuvant chemotherapy, with bleomycin, etoposide, and cisplatin (BEP), the patient received testosterone replacement therapy and remained free of recurrence at an 18-month follow-up., Conclusion: This case highlights both the rarity of a bilateral testicular tumor's synchronous appearance and its extremely infrequent discordant histopathology. A comprehensive review of the major series of SBTGCTs with discordant histology cited in the literature is additionally presented.

Background and Objectives : The impact of pure histological subtypes in testicular non-seminoma germ cell tumors on survival, specifically regarding pure embryonal carcinoma, is not well established. Therefore, this study aimed to test for differences between pure embryonal carcinoma and mixed germ cell tumor patients within stages I, II and III in a large population-based database. Materials and Methods : We relied on the Surveillance, Epidemiology and End Results (SEER) database (2004-2019) to identify testicular pure embryonal carcinoma vs. mixed germ cell tumor patients. Cumulative incidence plots depicted cancer-specific mortality that represented the main endpoint of interest. Multivariable competing risks regression models tested for differences between pure embryonal carcinoma and mixed germ cell tumor patients in analyses addressing cancer-specific mortality and adjusted for other-cause mortality. Results : Of 11,223 patients, 2473 (22%) had pure embryonal carcinoma. Pure embryonal carcinoma patients exhibited lower cancer-specific mortality relative to their mixed germ cell tumor counterparts for both stage III (13.9 vs. 19.4%; p < 0.01) and stage II (0.5 vs. 3.4%, p < 0.01), but not in stage I (0.9 vs. 1.6%, p = 0.1). In multivariable competing risks regression models, pure embryonal carcinoma exhibited more favorable cancer-specific mortality than mixed germ cell tumor in stage III (hazard ratio 0.71, p = 0.01) and stage II (hazard ratio 0.11, p < 0.01). Conclusions : Pure embryonal carcinoma exhibits a more favorable cancer-specific mortality profile relative to mixed germ cell tumor in stage II and III testicular cancers. Consequently, the presence of mixed germ cell tumor elements may be interpreted as a risk factor for cancer-specific survival.

Background: 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., Methods: 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., Results: 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., Conclusions: 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yao, Zhou, Duan, Wu, Li, Liu and Zhang.)

Germ cell neoplasia in situ (GCNIS) is the precursor of both seminomatous and non-seminomatous germ cell tumors. It consists of distended tubules that may have either intratubular seminoma or intratubular embryonal carcinoma cells. Many invasive non-seminomatous tumors contain a mixture of tumor types, which are reviewed here. Morphology, aided by a panel of immunostains, can determine the presence and percent of embryonal carcinoma, yolk sac tumor, choriocarcinoma, or teratoma in such tumors. Use of immunostains, required for diagnosis in perhaps 25% of testicular neoplasms, is reviewed. Changes of classification in the AJCC (8 th edition) in 2016 are discussed, including the partitioning of two tumor types: the central role of chromosome 12p amplification allows both teratoma and yolk sac tumor to be divided into prepubertal types (lacking amplification) and post-pubertal types. Occasionally, sex cord-stromal tumors, hematolymphoid tumors, or epididymal adenomatoid tumors enter the differential diagnosis of germ cell neoplasms., Competing Interests: Funding There are no sources of funding to report., (Copyright © 2022. Published by Elsevier Inc.)

Objective: To systematically evaluate evidence on prognostic factors for tumor recurrence in clinical stage I nonseminoma patients other than lymphovascular invasion (LVI)., Methods: We performed a systematic literature search in the biomedical databases Medline (via Ovid) and Cochrane Central Register of Controlled Trials (search period January 2010 to February 2021) for full text publications in English and German language, reporting on retro- or prospectively assessed prognostic factors for tumor recurrence in patients with stage I nonseminomatous germ cell tumors., Results: Our literature search yielded eleven studies reporting on 20 potential prognostic factors. Results are based on cohort studies of mostly moderate to low quality. Five out of eight studies found a significant association of embryonal carcinoma (EC) in the primary tumor with relapse. Among the different risk definitions of embryonal carcinoma (presence, predominance, pure), presence of EC alone seems to be sufficient for prognostification. Interesting results were found for rete testis invasion, predominant yolk sac tumor, T-stage and history of cryptorchidism, but the sparse data situation does not justify their clinical use., Conclusions: No additional factors that meet the prognostic value of LVI, especially when determined by immunohistochemistry, could be identified through our systematic search. The presence of EC might serve as a second, subordinate prognostic factor for clinical use as the data situation is less abundant than the one of LVI. Further efforts are necessary to optimize the use of these two prognostic factors and to evaluate and validate further potential factors with promising preliminary data., (© 2022. The Author(s).)

Background: Germ cell tumors (GCTs) are the most common type of cancer in Germany in young men between 15 and 44 years 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., Objectives: Updated understanding of the molecular processes underlying GCT development and their special forms as well as recommendations for new and useful biomarkers., Results: The development of GCTs is a dynamic process largely influenced by the microenvironment. Seminomas (SEs) in particular seem to posses a higher 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., Conclusion: 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., (© 2022. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Background: In recent years, several studies investigated the complex process called "reprogramming" of seminoma (S) cells. The accepted pathogenetic model is a complex network including SOX2, SOX17, OCT3/4 and PRAME, which modulates the epigenetic transcription of numerous downstream genes and drives a divergent gene expression profile resulting in the transition from pure S (P-S) to S component (S-C) of mixed germ cell tumors of the testis (M-GCTT), and finally to embryonal carcinoma (EC). Herein, we tested a large cohort of GCTT with SOX2 and PRAME to evaluate their expression in the evolutionary steps of GCTT and verify if the modulation in the expression of these two molecules could be relevant for the fate of GCTT., Methods: We tested 43, 19 and 17 consecutive and retrospectively enrolled cases of GCTT, germ cell neoplasia in situ (GCNIS) and uninvolved background testes (UBT), respectively. SOX2 and PRAME expressions have been evaluated with H-score and compared by adopting the appropriate statistic tests (Student's t-test and Mann-Whitney U test)., Results: We found that SOX2 was more expressed by nonseminomatous-GCTT (NS-GCTT) (p < 0.001) and EC (p < 0.001) rather than S; by contrast, PRAME showed an opposite expression profile being expressed by S but not by NS-GCTT (p < 0.001) and EC (p < 0.001). S-C showed different expressions of SOX2 and PRAME compared to both P-S (p = 0.002 and <0.001, respectively) and EC (p < 0.001 and 0.042, respectively), with intermediate values between these latter two categories. GCNIS and UBT showed no expression of SOX2 (scattered positive Leydig cells) but high H-score levels of PRAME., Conclusions: SOX2 and PRAME are differentially expressed and specularly modulated during the "reprogramming" of S cells [P-S (high levels of PRAME, no expression/low levels of SOX2) → S-C (intermediate levels of PRAME, intermediate levels of SOX2) → EC (no expression/low levels of PRAME, high levels of SOX2)], therefore supporting a complex pathogenetic model where the interactions between these two molecules are crucial in determining the fate of GCTT., Competing Interests: Declaration of Competing Interest All the Authors present in this article declare NO CONFLICT OF INTERESTS., (Copyright © 2022. Published by Elsevier GmbH.)

Introduction: Melanoma-associated antigen A2 (MAGE-A2) is a member of the cancer-testis antigen family differentially overexpressed in a variety of malignancies and is associated with tumor development. However, clinical significance and prognostic value of MAGE-A2 in different histological subtypes of testicular germ cell tumors (TGCTs) have not been explored., Materials and Methods: Here, we aimed to investigate the clinical significance and prognostic impact of MAGE-A2 expression in TGCTs compared to benign tumors as well as adjacent normal tissues and then between seminomas and non-seminomas groups using immunohistochemistry on tissue microarrays., Results: The results indicated a statistically significant difference between overexpression of MAGE-A2 and histological subtypes of TGCTs. A statistically significant association was found between a high level of nuclear expression of MAGE-A2 protein and advanced pT stage (P = 0.022), vascular invasion (P = 0.037), as well as involvement of rete testis (P = 0.022) in embryonal carcinomas. Increased nuclear expression of MAGE-A2 was observed to be associated with more aggressive behaviors and tumor progression rather than cytoplasmic expression in these cases. Further, high level nuclear expression of MAGE-A2 had shorter disease-specific survival (DSS) or progression-free survival (PFS) compared to patients with moderate and low expression of MAGE-A2, however, without a statistically significant association., Conclusion: Our results confirm that increased nuclear expression of MAGE-A2 has a clinical significance in embryonal carcinomas and is associated with progression of disease. Moreover, MAGE-A2 may act as a potential predictive biomarker for the prognosis in embryonal carcinomas if follow-up period becomes longer. Further investigations for the biological function of MAGE-A2 are required in future studies., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population-embryonal carcinoma (EC)-is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra-embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three-dimensional (3D) co-cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho- and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin-resistant subclones, highlighting CD24 as a putative target in combination with cisplatin., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Lymphovascular invasion (LVI) is a relevant prognostic factor in germ cell tumors of the testis (GCTT) and it has been included in the AJCC staging system. Nevertheless, its histological assessment is challenging, with low/moderate interobserver agreement also among expert uropathologists. Few studies focused on the potential role of immunohistochemistry to solve this critical issue; as result, in current guidelines there is no indication for additional staining to detect this histological feature. In the present study, we investigated the detection of LVI invasion in a small cohort of GCTT with double staining for OCT4/CD34. Although our results need to be validated in larger case series with follow-up data, they suggest as OCT4/CD34 could be a useful tool for the histological assessment of these tumors, helping to identify some histological mimickers of LVI and modifying the pT/stage in a significant percentage of patients., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Objective: To evaluate the perioperative and oncological outcomes after post-chemotherapy robot-assisted retroperitoneal lymph node dissection (PC-RARPLND)., Materials and Methods: We retrospectively reported the perioperative and oncological outcomes of all the patients with testicular cancer who underwent PC-RARPLND at three tertiary teaching centers. Descriptive statistical measures were used to report demographic, clinical, intraoperative, postoperative and oncological outcomes., Results: There were 43 consecutive patients who underwent PC-RARPLND at the participating institutions. Mean patient age was 29.2 years (± 8.2), BMI was 26.6 kg/m 2 (± 6.2). The mean size of retroperitoneal mass was 4.1 cm (± 3.5). Full bilateral template dissection was performed in 38 (88.3%) patients. Nerve sparing was attempted in 19 (44.1%) patients. Mean operative time was 374 min (± 132) and estimated blood loss was 292 ml (± 445.6). The mean postoperative LOS was 2.8 days (± 5.9). There was a total of 12 complications in 10 patients (Clavien grade I = 5, II = 3, III = 3 and IV = 1). Postoperative pathology demonstrated 24 patients (55%) with necrosis/fibrosis, 16 (37%) with teratoma and 3 (7%) with viable tumor. Mean lymph node (LN) yield was 26.5 LNs (SD ± 16.1). Patients were followed for a mean of 30.7 months (± 24.7). No deaths were documented during follow-up and 2 pulmonary recurrences were identified. Antegrade ejaculation was preserved in 70.6% of patient who underwent nerve sparing. Limitations included retrospective nature and limited follow up., Conclusion: PC-RAPLND is safe and technically reproducible. It provides improved morbidity and less convalescence., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

One hundred malignant mixed germ cell tumors of the ovary that occurred in patients 3 to 55 years (mean: 20 y) of age are described. The clinical presentation was usually that of any highly malignant tumor of the ovary (abdominal pain and distension), but rarely (3 cases) endocrine manifestations were present. The tumors were usually unilateral (96%), ranged from 4 to 38 cm (mean: 16 cm), and were uniformly solid or, more often, solid and cystic; occasionally the typical appearance of dysgerminoma could be appreciated. The most common tumor type was yolk sac tumor (91%), followed by dysgerminoma (61%), immature teratoma (58%), embryonal carcinoma (38%), and choriocarcinoma (11%). A variety of admixtures were encountered; dysgerminoma and yolk sac tumor was the most common combination (25% of the tumors) with the 2 components often being sharply demarcated. Immature teratoma and yolk sac tumor was the next most common pairing (20%) followed by yolk sac tumor and embryonal carcinoma, with or without immature teratoma (16%). Tumors with a choriocarcinoma component had the most varied combinations of tumor types. Embryoid bodies were seen in 21% of the tumors, most often as fragmented forms arranged in a nodular manner with yolk sac tumor and/or embryonal carcinoma; uncommonly they occurred singly or in clusters. Numerous confluent well-formed embryoid bodies (polyembryoma) were prominent in 2 tumors. Three tumors had a focal diffuse embryoma pattern. The specific tumor types showed the known diverse spectrum of microscopic appearances, but the frequent haphazard arrangement of 2 or more subtypes often resulted in complex morphology. Overgrowth of another neoplastic component, most often primitive neuroectodermal tumor, occurred in 10% of the tumors further complicating the histologic picture. This is the largest series of ovarian malignant mixed germ cell tumors reported and details their characteristics including associations of their subtypes and the frequent apparent role of embryoid bodies in giving rise to yolk sac tumor and embryonal carcinoma components., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Overactivation of the c-MET/HGF system is a feature of many cancers. We previously reported that type II testicular germ cell tumor (TGCT) cells express the c-MET receptor, forming non-seminomatous lesions that are more positive compared with seminomatous ones. Notably, we also demonstrated that NT2D1 non-seminomatous cells (derived from an embryonal carcinoma lesion) increase their proliferation, migration, and invasion in response to HGF. Herein, we report that HGF immunoreactivity is more evident in the microenvironment of embryonal carcinoma biopsies with respect to seminomatous ones, indicating a tumor-dependent modulation of the testicular niche. PI3K/AKT is one of the signaling pathways triggered by HGF through the c-MET activation cascade. Herein, we demonstrated that phospho-AKT increases in NT2D1 cells after HGF stimulation. Moreover, we found that this pathway is involved in HGF-dependent NT2D1 cell proliferation, migration, and invasion, since the co-administration of the PI3K inhibitor LY294002 together with HGF abrogates these responses. Notably, the inhibition of endogenous PI3K affects collective cell migration but does not influence proliferation or chemotactic activity. Surprisingly, LY294002 administered without the co-administration of HGF increases cell invasion at levels comparable to the HGF-administered samples. This paradoxical result highlights the role of the testicular microenvironment in the modulation of cellular responses and stimulates the study of the testicular secretome in cancer lesions.

Background: Testicular germ cell tumors (TGCTs) are the most common malignant cancer in young men. Although TGCTs are generally responsive to platinum-based chemotherapy particularly cisplatin, acquired resistance in patients with metastasis still occurs resulting in poor prognosis. Specifically, differentiation of embryonal carcinoma (EC) cells, the stem cells of TGCTs, can lead to the reduction of cisplatin responsiveness. Therefore, novel therapeutic strategies for TGCTs are needed. System L amino acid transporters have been reported to be up-regulated and to play an important role in tumorigenesis. However, expression and role of system L amino acid transporters in TGCTs remain elusive., Materials and Methods: Expression of system L amino acid transporters was analyzed in TGCT samples from The Cancer Genome Atlas (TCGA). Expression of LAT1, LAT2, and 4F2hc was examined in human embryonal carcinoma cell line NTERA2. Roles of system L amino acid transporters on NTERA2 cell survival, cell proliferation, pluripotency, and cisplatin sensitivity were evaluated., Results: Based upon TCGA datasets, we found that two isoforms of system L (LAT1 and LAT2) and their chaperone protein 4F2hc are highly expressed in EC samples compared with other groups. Treatment with the system L inhibitor BCH significantly suppressed leucine uptake into the pluripotent EC cell line NTERA2. The malignant phenotypes including cell viability, cell proliferation, and clonal ability were decreased following BCH treatment. Nonetheless, system L inhibition did not alter expression of stemness genes in NTERA2 cells. After NTERA2 differentiation, expressions of LAT1 and LAT2 were decreased. Finally, co-administration of BCH enhanced cisplatin sensitivity in both undifferentiated and differentiated cells. These effects were associated with the reduction in p70S6K phosphorylation., Conclusion: Taken together, these results shed light on the roles of system L amino acid transporters in TGCTs. Therefore, system L amino acid transporters could provide novel therapeutic targets for treatment against TGCTs., (© 2020 American Society of Andrology and European Academy of Andrology.)

Introduction: hTERT (human telomerase reverse transcriptase) is a catalytic subunit of the enzyme telomerase and has a role in cell proliferation, cellular senescence, and human aging., Materials and Methods: The purpose of this study was to evaluate the expression and significance of hTERT protein expression as a prognostic marker in different histological subtypes of testicular germ cell tumors (TGCTs), including 46 embryonal carcinomas, 46 yolk sac tumors, 38 teratomas, 84 seminomas as well as two main subtypes of seminomas and non-seminomas using tissue microarray (TMA) technique., Results: The results showed that there is a statistically significance difference between the expression of hTERT and various histological subtypes of TGCTs (P < 0.001). In embryonal carcinoma, low level expression of hTERT protein was significantly associated with advanced pT stage (P = 0.023) as well as tunica vaginalis invasion (P = 0.043). Moreover, low level expression of hTERT protein was found to be a significant predictor of worse DSS (log rank: P = 0.011) and PFS (log rank: P = 0.011) in the univariate analysis. Additionally, significant differences were observed (P =0.021, P =0.018) with 5-year survival rates for DSS and PFS of 66% and 70% for moderate as compared to 97% and 97% for high hTERT protein expression, respectively., Conclusion: We showed that hTERT protein expression was associated with more aggressive tumor behavior in embryonal carcinoma patients. Also, hTERT may be a novel worse prognostic indicator of DSS or PFS, if the patients are followed up for more time periods.

Hepatocyte Nuclear Factor 1 beta (HNF1β) is a transcription factor which plays an important role during early organogenesis, especially of the pancreato-biliary and urogenital tract. Furthermore, HNF1β is an established marker in the differential diagnosis of ovarian cancer and shows a distinct nuclear expression in the clear cell carcinoma subtype. Recently, it has been described in yolk sac tumor, which represents a common component in many non-seminomatous germ cell tumors. Due to its broad histologic diversity, the diagnosis may be challenging and additional tools are very helpful in the workup of germ cell tumors. Immunohistochemistry was used to study HNF1β expression in a tissue microarray (TMA) of 601 testicular germ cell tumors including seminoma, embryonal carcinoma, yolk sac tumor, choriocarcinoma, teratoma, germ cell neoplasia in situ (GCNIS), and normal tissue. The expression pattern was compared to glypican 3 (GPC3) and α-fetoprotein (AFP), two markers currently in use for the detection of yolk sac tumor. HNF1β showed a distinct nuclear staining in comparison to the cytoplasmic pattern of GPC3 and AFP. The sensitivity and specificity of HNF1β were 85.4% and 96.5%, of GPC3 83.3% and 90.7%, of AFP 62.5% and 97.7%. We conclude that HNF1β allows a reliable distinction of yolk sac tumor from other germ cell tumor components. Therefore, we propose HNF1β as a novel and robust marker in the immunohistochemical workup of testicular germ cell tumors.

The pluripotent mouse embryonal carcinoma cell line P19 is widely used as a model for research on all-trans-retinoid acid (RA)-induced neuronal differentiation; however, the signaling pathways involved in this process remain unclear. This study aimed to reveal the molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells. Real-time quantitative polymerase chain reaction and Western blot analysis were used to determine the expression of neuronal-specific markers, whereas flow cytometry was used to analyze cell cycle and cell apoptosis. The expression profiles of messenger RNAs (mRNAs) in RA-induced neuronal differentiation of P19 cells were analyzed using high-throughput sequencing, and the functions of differentially expressed mRNAs (DEMs) were determined by bioinformatics analysis. RA induced an increase in both class III β-tubulin (TUBB3) and neurofilament medium (NEFM) mRNA expression, indicating that RA successfully induces neuronal differentiation of P19 cells. Cell apoptosis was not affected; however, cell proliferation decreased. We found 4117 DEMs, which were enriched in the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, Wnt signaling pathway, and cell cycle. Particularly, a few DEMs could be identified in the PI3K/Akt signaling pathway networks, such as PI3K, Akt, glycogen synthase kinase-3β (GSK3β), cyclin-dependent kinase 4 (CDK4), P21, and Bax. RA significantly increased the protein expression of PI3K, Akt, phosphorylated Akt, GSK3β, phosphorylated GSK3β, CDK4, and P21, but it reduced Bax protein expression. The Akt inhibitor affected the increase of TUBB3 and NEFM mRNA expression in RA-induced P19 cells. The molecular mechanism underlying the RA-induced neuronal differentiation of P19 cells is potentially involved in the PI3K/Akt/GSK3β signaling pathway. The decreased cell proliferation ability of neuronally differentiated P19 cells could be associated with the expression of cell cycle proteins., (© 2020 Wiley Periodicals, Inc.)

Background and Importance: Primary tumors of the brachial plexus are rare. Most are benign and characterized as Schwannoma and neurofibroma, whereas malignant peripheral nerve sheath tumors are less common. Here, we report a rare case of primary embryonal carcinoma in the brachial plexus., Clinical Presentation: A 17-yr-old male presented with a 3-mo history of a mass growing in the left supraclavicular region over the middle part of the clavicle. Magnetic resonance imaging revealed a well-defined mass (diameter 2.5 cm) straddling the brachial plexus. After surgical resection, and the mass was histologically confirmed to be an embryonal carcinoma., Conclusion: Primary embryonal carcinoma in the brachial plexus has not been reported previously. This case highlights the importance of considering the possibility that some primary brachial plexus tumors may be malignant and should be treated promptly., (Copyright © 2019 by the Congress of Neurological Surgeons.)

The pharmacological effects of BST204-a fermented ginseng extract-on several types of cancers have been reported. However, the effects of ginseng products or single ginsenosides against cancer stem cells are still poorly understood. In this study, we identified the anti-tumorigenic and anti-invasive activities of BST204 through the suppression of the cancer stem cell marker, CD133. The treatment of embryonic carcinoma cells with BST204 induced the expression of the tumor suppressor protein, p53, which decreased the expression of cell cycle regulatory proteins and downregulated the expression of CD133 and several stemness transcription factors. These changes resulted in both the inhibition of tumor cell proliferation and tumorigenesis. The knockdown of CD133 suggests that it has a role in tumorigenesis, but not in cancer cell proliferation or cell cycle arrest. Treatment with BST204 resulted in the reduced expression of the mesenchymal marker, N-cadherin, and the increased expression of the epithelial marker, E-cadherin, leading to the suppression of tumor cell migration and invasion. The knockdown of CD133 also exhibited an anti-invasive effect, indicating the role of CD133 in tumor invasion. The single ginsenosides Rg3 and Rh2-major components of BST204-exhibited limited effects against cancer stem cells compared to BST204, suggesting possible synergism among several ginsenoside compounds.

Testicular carcinoma recurrences represent a rare finding (1-6% in non-seminomatous germ cell tumours). However, cases of recurrence have been described many years later. We report a case of late recurrence of embryonic testicular carcinoma, after 26 years, with pulmonary metastases. Following evidence of increase of alpha-fetoprotein (AFP), the patient underwent a total body computed tomography scan that exhibited two pulmonary nodules, one in upper left lobe and other in left hilar region with multiple mediastinal and retrocrural lymph node enlargements All consolidations showed increased sugar uptake value at PET CT. Biopsies of lung consolidations confirmed diagnosis of recurrence of testicular carcinoma.

Embryonal carcinomas (ECs) and seminomas are testicular germ cell tumors. ECs display expression of SOX2, while seminomas display expression of SOX17. In somatic differentiation, SOX17 drives endodermal cell fate. However, seminomas lack expression of endoderm markers, but show features of pluripotency. Here, we use chromatin immunoprecipitation sequencing to report and compare the binding pattern of SOX17 in seminoma-like TCam-2 cells to SOX17 in somatic cells and SOX2 in EC-like 2102EP cells. In seminoma-like cells, SOX17 was detected at canonical (SOX2/OCT4), compressed (SOX17/OCT4) and noncomposite SOX motifs. SOX17 regulates TFAP2C, PRDM1 and PRDM14, thereby maintaining latent pluripotency and suppressing somatic differentiation. In contrast, in somatic cells canonical motifs are rarely bound by SOX17. In sum, only 12% of SOX17-binding sites overlap in seminoma-like and somatic cells. This illustrates that binding site choice is highly dynamic and cell type specific. Deletion of SOX17 in seminoma-like cells resulted in loss of pluripotency, marked by a reduction of OCT4 protein level and loss of alkaline phosphatase activity. Furthermore, we found that in EC-like cells SOX2 regulates pluripotency-associated genes, most likely by partnering with OCT4. In conclusion, SOX17 (in seminomas) functionally replaces SOX2 (in ECs) to maintain expression of the pluripotency cluster., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Objective: To systematically review the literature on the prognostic value of lymphovascular invasion (LVI) and embryonal carcinoma (EC) for occult metastatic disease in clinical stage I nonseminomatous germ cell tumour (CS I NSGCT)., Materials and Methods: The PubMed, Embase (OVID) and SCOPUS databases were searched up to March 2019. Studies reporting on the association between LVI and/or EC and occult metastatic disease were considered for inclusion. The quality and risk of bias were evaluated by the Quality in Prognosis Studies tool., Results: We screened 5287 abstracts and 207 full-text articles. We included 35 studies in the narrative synthesis and 24 studies in a meta-analysis. LVI showed the strongest effect. Pooled rates of occult metastasis were 47.5% and 16.9% for LVI-positive and LVI-negative patients, respectively (odds ratio [OR] 4.33, 95% confidence interval [CI] 3.55-5.30; P < 0.001). Pooled rates of occult metastasis were 33.2% for EC presence and 16.2% for EC absence (OR 2.49, 95% CI 1.64-3.77; P < 0.001). Pooled rates of occult metastasis were 40.0% for EC >50% and 20.0% for EC <50% (OR 2.62, 95% CI 1.93-3.56; P < 0.001)., Conclusions: LVI is the strongest risk factor for relapse. The prognostic value of EC is high, but there is no common agreement on how to define this risk factor. Both EC presence and EC >50% have similar ORs for occult metastasis. This shows that the assessment of EC presence is sufficient for the classification of EC., (© 2019 The Authors. BJU International Published by John Wiley & Sons Ltd on behalf of BJU International.)

An aberrant increase in pluripotency gene (PpG) expression due to enhancer reactivation could induce stemness and enhance the tumorigenicity of cancer stem cells. Silencing of PpG enhancers (PpGe) during embryonic stem cell differentiation involves Lsd1-mediated H3K4me1 demethylation and DNA methylation. Here, we observed retention of H3K4me1 and DNA hypomethylation at PpGe associated with a partial repression of PpGs in F9 embryonal carcinoma cells (ECCs) post-differentiation. H3K4me1 demethylation in F9 ECCs could not be rescued by Lsd1 overexpression. Given our observation that H3K4me1 demethylation is accompanied by strong Oct4 repression in P19 ECCs, we tested if Oct4 interaction with Lsd1 affects its catalytic activity. Our data show a dose-dependent inhibition of Lsd1 activity by Oct4 and retention of H3K4me1 at PpGe in Oct4-overexpressing P19 ECCs. These data suggest that Lsd1-Oct4 interaction in cancer stem cells could establish a "primed" enhancer state that is susceptible to reactivation, leading to aberrant PpG expression., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Germ cell neoplasia in situ is the initial manifestation for invasive germ cell tumor. Further progression will result in intratubular germ cell tumor with the majority being intratubular seminoma or intratubular embryonal carcinoma. Intratubular teratoma in the testis is exceptionally rare with no well-documented cases to our knowledge. In this article, we report a case of an intratubular teratoma adjacent to mixed germ cell tumor in the testis. The patient is a 34-year-old male who presented with a palpable right testicular mass and underwent right radical orchiectomy. Gross examination of the testis revealed 2.0-cm tan, well-circumscribed, firm, and nodular mass at the inferior pole. Microscopic examination revealed a mixed germ cell tumor, predominantly seminoma (95%) with embryonal carcinoma (4%) and teratoma (1%). There is also germ cell neoplasia in situ, intratubular seminoma, and intratubular teratoma at the periphery of the tumor. Tubules with intratubular teratoma were filled by neoplastic squamous cells with a single layer of germ cell neoplasia in situ at the periphery. Adjacent to the intratubular teratoma was seminoma, embryonal carcinoma, and invasive teratoma. Immunohistochemical stains showed the neoplastic squamous cells in the tubule to be positive for p40 and negative for OCT34 and D2-40. The single layer of germ cell neoplasia in situ at the periphery of the intratubular teratoma was negative for p40 and positive for OCT34 and D2-40. Although teratoma is a common component in an adult germ cell tumor, an intratubular manifestation is exceptional. The present case illustrates this rare finding.

Purpose: Active surveillance (AS) for testicular nonseminomatous germ cell tumors (NSGCT) is widely used. Although there is no consensus for optimal treatment at relapse on surveillance, globally patients typically receive chemotherapy. We describe treatment of relapses in our non-risk-adapted NSGCT AS cohort and highlight selective use of primary retroperitoneal lymph node dissection (RPLND)., Methods: From December 1980 to December 2015, 580 patients with clinical stage I NSGCT were treated with AS, and 162 subsequently relapsed. First-line treatment was based on relapse site and extent. Logistic regression was used to explore factors associated with need for multimodal therapy on AS relapse., Results: Median time to relapse was 7.4 months. The majority of relapses were confined to the retroperitoneum (66%). After relapse, first-line treatment was chemotherapy for 95 (58.6%) and RPLND for 62 (38.3%), and five patients (3.1%) underwent other therapy. In 103 (65.6%), only one modality of treatment was required: chemotherapy only in 58 of 95 (61%) and RPLND only in 45 of 62 (73%). Factors associated with multimodal relapse therapy were larger node size (odds ratio, 2.68; P = .045) in patients undergoing chemotherapy and elevated tumor markers (odds ratio, 6.05; P = .008) in patients undergoing RPLND. When RPLND was performed with normal markers, 82% required no further treatment. Second relapse occurred in 30 of 162 patients (18.5%). With median follow-up of 7.6 years, there were five deaths (3.1% of AS relapses, but 0.8% of whole AS cohort) from NSGCT or treatment complications., Conclusion: The retroperitoneum is the most common site of relapse in clinical stage I NSGCT on AS. Most are cured by single-modality treatment. RPLND should be considered for relapsed patients, especially those with disease limited to the retroperitoneum and normal markers, as an option to avoid chemotherapy.

The protein phosphatase PPM1D (Wip1) was originally identified as a p53 target product. Activation of PPM1D through various mechanism promotes the tumorigenic potential of various cancers by suppressing p53 and other DNA damage response proteins. New functions of PPM1D have recently been revealed in physiological processes such as cell differentiation. However, the regulatory mechanisms of signalling pathway to maintain stemness and induce cell differentiation are still unclear. Here we report that PPM1D modulates retinoic acid (RA) signalling. PPM1D knockdown resulted in decreased alkaline phosphatase activity of the human teratocarcinoma cell line NT2/D1. Inhibition of PPM1D-induced cell differentiation and decreased gene expression of the stem cell marker Oct-4 (POU5F1). RA-induced cell differentiation was promoted by reducing PPM1D activity. RA treatment elicited activation of the MEK-ERK pathway and induced rapid and transient activation of the extracellular signal-regulated kinase 1/2 (ERK-1/2). PPM1D dephosphorylated a phosphopeptide with the TEY motif in ERK-1/2 in vitro. Moreover, phosphorylation of ERK-1/2 was facilitated by PPM1D inhibition. Our study shows that PPM1D plays an important role in maintaining the undifferentiation state and a new function in RA-induced ERK regulation and cell differentiation., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

In 2016, the WHO classification of testicular germ cell tumors was revised considering advances in the understanding of their tumorigenesis and molecular features. This restructuring led to a division into two major groups with, on one hand, prepubertal-type tumors, not derived from germ cell neoplasia in situ (GCNIS), and on the other hand, postpubertal-type tumors, GCNIS-derived, which occur in youg men (seminoma and non seminomatous germ cell tumors - embryonal carcinoma, yolk sac tumor, teratoma and choriocarcinoma essentially). The term germ cell neoplasia in situ is consensually accepted as a new terminology for the precursor lesion. In this new classification, the term "spermatocytic seminoma" is replaced by "spermatocytic tumor", reclassified among non-GCNIS-derived tumors. The purpose of this change of nomenclature is to reflect the usually non-aggressive behaviour of this tumor and to avoid any confusion with usual seminoma. The spectrum of trophoblastic tumors continues to expand with the description of new rare entities such as the cystic trophoblastic tumor, the placental site trophoblastic tumor and the epithelioid trophoblastic tumor. This review aims to provide a focus on testicular germ cell tumors highlighting the new immunohistochemical and molecular features responsible for the restructuring of classification. The TNM staging is presented according to the AJCC 8th edition 2017 update., (Copyright © 2019 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Spermatogenesis is a complex cell differentiation process that includes marked genetic, cellular, functional and structural changes. It requires tight regulation, because disturbances in any of the spermatogenic processes would lead to fertility deficiencies as well as disorders in offspring. To increase our knowledge of signal transduction during sperm development, we carried out a large-scale identification of the phosphorylation events that occur in the human male gonad. Metal oxide affinity chromatography using TiO 2 combined with LC-MS/MS was conducted to profile the phosphoproteome of adult human testes with full spermatogenesis. A total of 8187 phosphopeptides derived from 2661 proteins were identified, resulting in the most complete report of human testicular phosphoproteins to date. Phosphorylation events were enriched in proteins functionally related to spermatogenesis, as well as to highly active processes in the male gonad, such as transcriptional and translational regulation, cytoskeleton organization, DNA packaging, cell cycle and apoptosis. Moreover, 174 phosphorylated kinases were identified. The most active human protein kinases in the testis were predicted both by the number of phosphopeptide spectra identified and the phosphorylation status of the kinase activation loop. The potential function of cyclin-dependent kinase 12 (CDK12) and p21-activated kinase 4 (PAK4) has been explored by in silico , protein-protein interaction analysis, immunodetection in testicular tissue, and a functional assay in a human embryonal carcinoma cell line. The colocalization of CDK12 with Golgi markers suggests a potential crucial role of this protein kinase during sperm formation. PAK4 has been found expressed in human spermatogonia, and a role in embryonal carcinoma cell response to apoptosis has been observed. Together, our protein discovery analysis confirms that phosphoregulation by protein kinases is highly active in sperm differentiation and opens a window to detailed characterization and validation of potential targets for the development of drugs modulating male fertility and tumor behavior., (© 2019 Castillo et al.)

The paternally-imprinted genes insulin-like growth factor 2 (IGF2), H19, delta-like homologue 1 (DLK1), and maternally-expressed gene 3 (MEG3) are expressed from the tandem gene loci IGF2-H19 and DLK1-MEG3, which play crucial roles in initiating embryogenesis and development. The erasure of imprinting (EOI) at differentially methylated regions (DMRs) which regulate the expression of these genes maintains the developmental quiescence of primordial germ cells (PGCs) migrating through the embryo proper during embryogenesis and prevents them from forming teratomas. To address the potential involvement of the IGF2-H19 and DLK1-MEG3 loci in the pathogenesis of embryonal carcinoma (EC), we investigated their genomic imprinting at DMRs in the human PGC-derived EC cell line NTera-2 (NT2). We observed EOI at the IGF2-H19 locus and, somewhat to our surprise, a loss of imprinting (LOI) at the DLK1-MEG3 locus. As a result, NT2 cells express imprinted gene ratios from these loci such that there are i) low levels of the proliferation-promoting IGF2 relative to ii) high levels of the proliferation-inhibiting long noncoding RNA (lncRNA) H19 and iii) high levels of proliferation-promoting DLK1 relative to iv) low levels of the proliferation-inhibiting lncRNA MEG3. Consistent with this pattern of expression, the knockdown of DLK1 mRNA by shRNA resulted in decreased in vitro cell proliferation and in vivo tumor growth as well as decreased in vivo organ seeding by NT2 cells. Furthermore, treatment of NT2 cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-azaD) inhibited their proliferation. This inhibition was accompanied by changes in expression of both tandem gene sets: a decrease in the expression of DLK1 and upregulation of the proliferation-inhibiting lncRNA MEG3, and at the same time upregulation of IGF2 and downregulation of the lncRNA H19. These results suggest that the DLK1-MEG3 locus, and not the IGF2-H19 locus, drives the tumorigenicity of NT2 cells. Based on these results, we identified DLK1 as a novel treatment target for EC that could be downregulated by 5-azaD.

Identification of the yolk sac tumor (YST) component in germ cell tumors (GCT) may prove challenging, and highly sensitive and specific immunohistochemical markers are still lacking. Preliminary data from the literature suggest that HNF1β may represent a sensitive marker of YST. The specificity of HNF1β has not been addressed in GCT. A cohort of 49 YST specimens from 45 patients was designed, occurring either as pure tumors, or as a component of a mixed GCT. Immunohistochemistry was conducted on whole tumor sections using HNF1β. SALL4, OCT4, CD30, CDX2, Cytokeratin 19, Glypican 3, and GATA3 were used for classification of the GCT components. Patients were mostly male (39/45), aged 14 months to 49 years, with primary testicular tumors (37/39), or primary mediastinal pure YSTs (2/39). All 6 primary tumors occurring in females (6/45) were pure ovarian YSTs; age range was 4 to 72 years. HNF1β nuclear reactivity was seen in the YST component in all 49 tumors, with a moderate to strong nuclear pattern of staining. Embryonal carcinoma (EC, 0/32) and seminoma (0/6) were negative. Choriocarcinoma (6/6) showed faint focal cytoplasmic reactivity to HNF1β but no nuclear staining. In teratomas, only enteric-type glands showed nuclear reactivity to HNF1β (11/16). Therefore, HNF1β sensitivity in YST component identification was 100% and specificity was 80%. Thus, in our experience, HNF1β is a sensitive and reliable marker of the YST component in GCT, and allows distinction of YST from intricately admixed EC, especially in the diffuse embryoma pattern., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cancer cells with a less differentiated stem-like phenotype are more resistant to therapeutic manipulations than their differentiated counterparts, and are considered as one of the main causes of cancer persistence and relapse. As such, induction of differentiation in cancer stem-like cells (CSLCs) has emerged as an alternative strategy to enhance the efficacy of anticancer therapies. CSLCs are metabolically distinct from differentiated cells, and any aberration from the intrinsic metabolic state can induce differentiation of CSLCs. Therefore, metabolism-related molecular targets, with a capacity to promote differentiation within CSLCs, are of therapeutic importance. Here, we demonstrate that phosphoglycerate dehydrogenase (PHGDH), an essential enzyme catalyzing the synthesis of amino acid serine, is important for maintaining the poorly differentiated, stem-like state of CSLCs. Our data shows that PHGDH deficiency impairs the tumorsphere formation capacity in embryonal carcinoma stem-like cells (ECSLCs), breast cancer stem-like cells (BCSLCs) and patient-derived brain tumor-initiating cells (BTICs), which is accompanied by the reduced expression of characteristic stemness-promoting factors, such as Oct4, Nanog, Sox-2, and Bmi-1. Mechanistically, PHGDH deficiency in ECSLCs promotes differentiation to various lineages via degradation of Oct4 and by increasing the stability of differentiation marker β3-tubulin. Furthermore, PHGDH inhibition promotes p-mTOR independent but Beclin-1-dependent autophagy, independent of apoptosis. When studied in combination, the inhibition of both PHGDH and p-mTOR in ECSLCs causes further augmentation of autophagy, and additionally promotes apoptosis, demonstrating the clinical applicability of PHGDH-based manipulations in cancer therapies. Recapitulating these in vitro findings in CSLC models, the intratumoral PHGDH expression in patient-derived tumors is positively correlated with the mRNA levels of stemness factors, especially Oct4, and cancer patients co-expressing high levels of PHGDH and Oct4 display significantly lower survival than those with low PHGDH/Oct4 co-expression. Altogether, this study identifies a clinically-relevant role for PHGDH in the regulation of stemness-differentiation axis within CSLCs.

The ability to self-renew is one of the most important properties of embryonic stem (ES) cells. Pluripotin (SC1), a small molecule with high activity and low toxicity, promotes self-renewal in mouse ES cells. SC1 can noticeably change the morphology of retinoic acid (RA)-induced F9 embryonic carcinoma cells (F9 cells). However, in the long term, RA and SC1 together cause cell apoptosis. When being added after 18-24 h of RA-induced F9 cell differentiation, SC1 transitorily activated Nanog and Oct4. Both Nanog and Oct4 were downregulated when SC1 and RA were added simultaneously. On the other hand, Klf4 was continually activated when SC1 was added between 6 and 24 h. Phosphorylated Erk1/2 protein levels were reduced from 6 to 24 h, whereas unphosphorylated Erk1 protein levels remained unchanged. A higher concentration of SC1 promoted cell self-renewal by strengthening the inhibition of Erk1/2 protein phosphorylation in F9 cells. Furthermore, SC1 and RA affect global DNA methylation by influencing the expressions of methylation-associated proteins, including Dnmt3b, Dnmt3l, Tet1, Tet2, and Tet3. In conclusion, SC1 inhibits the differentiation of RA-induced F9 cells mainly by reducing the levels of phosphorylated Erk1/2 and enhancing the expression of Klf4, although it also reduces DNA methylation, which may have an additional effect on ES cell differentiation.

Tumors with a neuronal component comprise a small percentage of central nervous system (CNS) neoplasms overall, but the presence of neuronal differentiation has important diagnostic, prognostic, and therapeutic implications. Insulinoma-associated protein 1 (INSM1) is a transcription factor with strong nuclear immunostaining in neuroendocrine cells and neoplasms of neuroendocrine origin; however, its expression in the CNS in normal brain and in neoplastic cells has not been fully explored. Here, we present immunostaining results from a large number of archival CNS tissue specimens, including 416 tumors. Nuclear immunostaining for INSM1 was frequently seen in medulloblastomas (87%, n = 94). Diffuse nuclear INSM1 immunostaining was detected in all central neurocytomas and pituitary adenomas. Patchy to rare staining with INSM1 was also seen in other high-grade embryonal tumors and high-grade gliomas. In normal brain tissue, specific nuclear INSM1 immunohistochemical staining was only seen in early neuronal development. Notably, nuclear INSM1 staining was not seen in adult normal brain, including areas of gliosis. These findings indicate that nuclear INSM1 immunostaining may serve as a strong nuclear marker in the brain for neoplasms of neuroendocrine or immature neuronal differentiation, when used in concert with other immunostains.

Leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1) is implicated in the regulation of signal transduction, transcription, RNA processing and tumor development. However, LRWD1 transcriptional regulation is not fully understood. This study aimed to investigate the relationship between LRWD1 expression and reactive oxygen species (ROS) level in human embryonal carcinoma cell line, NT2/D1 cells, which will help in understanding the transcriptional regulatory role of ROS in cells. Results showed that the exposure of NT2/D1 cells to various concentrations of hydrogen peroxide (H 2 O 2 ) and the nitric oxide (NO) donor sodium nitroprusside (SNP) caused a significant increase in the mRNA and protein expression of LRWD1. In addition, LRWD1 promoter luciferase reporter assay, and Chromatin Immunoprecipitation assay (CHIP assay) showed that nuclear factor erythroid-2-related factor (Nrf2) was involved in the regulation of LRWD1 expression in response to oxidative stress. The involvement of Nrf2 was confirmed by shRNA-mediated knockdown of Nrf2 in NT2/D1 cells, which caused a significant decrease in LRWD1 expression in response to oxidative stress. Similarly, LRWD1 knockdown resulted in the accumulation of H 2 O 2 and superoxide anion radical (O2-). Blocking ROS production by N-acetyl cysteine (NAC) protected NT2/D1 shLRWD1cells from H 2 O 2 -induced cell death. Collectively, oxidative stress increased LRWD1 expression through a Nrf2-dependent mechanism, which plays an important role in cellular adaptation to oxidative stress. These results highlight an evidence, on the molecular level, about LRWD1 transcriptional regulation under oxidative stress., (Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

We demonstrate that OCT4 expression is regulated by germ cell nuclear factor (GCNF) via its interactions with three nuclear receptor (NR) binding sites within OCT4 promoter conserved regions (CRs) in human embryonic carcinoma (EC) NCCIT cells. OCT4 expression is gradually reduced during the retinoic acid-induced differentiation, while GCNF temporarily increased after 2 days and then significantly decreased. In addition, OCT4 expression is significantly reduced by overexpression of exogenous GCNF, but increased by GCNF shRNA-mediated knockdown. The transcriptional activity of OCT4 is significantly inhibited by dose-dependent overexpression of GCNF. While mutants at each of the NR binding sites retain the repressive effects of GCNF on OCT4 promoter activity, the repressive effect was completely eliminated in the reporter construct with all binding sites mutated even in the presence of GCNF. Furthermore, the transcriptional activity of native minimal promoter (CR1-Luc) containing the first NR binding site was significantly reduced by GCNF overexpression, while the mutant retained basal activity to some extent. Next, an exogenous minimal ti promoter-inserted CR2 reporter construct containing the second and third NR binding sites (CR2-ti-Luc) was co-transfected with GCNF expression vector. The transcriptional activity of CR2-ti-Luc was significantly decreased by GCNF overexpression, while mutation of both binding sites retained the transcriptional activity of the reporter construct. Binding assays confirmed the direct interaction of GCNF with all three NR binding sites cooperatively. Taken together, GCNF acts as a transcriptional repressor in the regulation of OCT4 gene expression through cooperative interaction with three NR binding elements in pluripotent NCCIT cells., (© 2017 Wiley Periodicals, Inc.)

Leucine-rich repeats and WD repeat domain containing protein 1 (LRWD1) is a testis-specific protein that mainly expressed in the sperm neck where centrosome is located. By using microarray analysis, LRWD1 is identified as a putative gene that involved in spermatogenesis. However, its role in human male germ cell development has not been extensively studied. When checking in the semen of patients with asthenozoospermia, teratozoospermia, and asthenoteratozoospermia, the level of LRWD1 in the sperm neck was significantly reduced with a defective neck or tail. When checking the sub-cellular localization of LRWD1 in the cells, we found that LRWD1 resided in the centrosome and its centrosomal residency was independent of microtubule transportation in NT2/D1, the human testicular embryonic carcinoma, cell line. Depletion of LRWD1 did not induce centrosome re-duplication but inhibited microtubule nucleation. In addition, the G1 arrest were observed in LRWD1 deficient NT2/D1 cells. Upon LRWD1 depletion, the levels of cyclin E, A, and phosphorylated CDK2, were reduced. Overexpression of LRWD1 promoted cell proliferation in NT2/D1, HeLa, and 239T cell lines. In addition, we also observed that autophagy was activated in LRWD1 deficient cells and inhibition of autophagy by chloroquine or bafilomycin A1 promoted cell death when LRWD1 was depleted. Thus, we found a novel function of LRWD1 in controlling microtubule nucleation and cell cycle progression in the human testicular embryonic carcinoma cells. J. Cell. Biochem. 119: 314-326, 2018. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Statins are a class of drugs that inhibit the rate-limiting steps in the cholesterol biosynthesis pathway. They act by inhibiting 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, which catalyzes the conversion of HMG-CoA to mevalonate. Blocking of mevalonate synthesis leads to inhibition of the farnesylation and geranylgeranylation of several functional proteins, such as RhoA and other small guanosine triphosphate-binding proteins, that are important in maintaining the undifferentiated status of the cells. In the present study, we hypothesized that simvastatin, likely through the inhibition of farnesylation and geranylgeranylation of Rac1, Cd42 and RhoA, induces a destruction/restructuration of the cytoskeleton that decreases mechanical strain transfer to the nuclei, inducing the loss of transmission of regulatory signals from the cytoskeleton to the nucleoskeleton. Although this remains at present a hypothesis and is not easy to define if the de-structuration of the cytoskeleton is a secondary effect of simvastatin treatment or the inhibition of post-translational protein modification have a precise role in the structuration of actin cytoskeleton, we speculate that these signal variations could inhibit the expression of certain stemness genes, which could therefore be considered nucleoskeleton-associated and mechanically regulated genes. On the other hand, the restructuration of the cytoskeleton inhibits the formation of lamellipodia and filopodia, which likely decreases the capability of cancer cells to invade the extracellular matrix, thereby modulating the equilibrium between proliferation, differentiation and metastatic invasion in human cancer cells. On the basis of our results we think that simvastatin, alone or in combination with conventional drugs, may have a possible role in cancer therapy.

Immune surveillance is a critical component of the antitumor response in vivo , yet the specific components of the immune system involved in this regulatory response remain unclear. In this study, we demonstrate that autoantibodies can mitigate tumor growth in vitro and in vivo We generated two cancer cell lines, embryonal carcinoma and glioblastoma cell lines, from monkey-induced pluripotent stem cells (iPSC) carrying a homozygous haplotype of major histocompatibility complex (MHC, Mafa in Macaca fascicularis). To establish a monkey cancer model, we transplanted these cells into monkeys carrying the matched Mafa haplotype in one of the chromosomes. Neither Mafa-homozygous cancer cell line grew in monkeys carrying the matched Mafa haplotype heterozygously. We detected in the plasma of these monkeys an IgG autoantibody against GRP94, a heat shock protein. Injection of the plasma prevented growth of the tumor cells in immunodeficient mice, whereas plasma IgG depleted of GRP94 IgG exhibited reduced killing activity against cancer cells in vitro These results indicate that humoral immunity, including autoantibodies against GRP94, plays a role in cancer immune surveillance. Cancer Res; 77(21); 6001-10. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Purpose: Bronchial brushings (BB) commonly aid in the diagnosis of primary lung cancer. However, the utility of this method in diagnosing endobronchial metastases (EBM) from extrapulmonic malignancies has not been thoroughly evaluated. The purpose of this study is to evaluate the sensitivity of BB in diagnosing EBM., Methods: An institutional database was queried for all patients with cytologically or histologically confirmed extrapulmonary EBM identified by endobronchial biopsy between 1978 and 2013. Data were collected on patient demographics, histologic and cytologic diagnoses, time from primary malignancy to identification of EBM, and location of EBM. The sensitivity of BB for the diagnosis of EBM and the clinicopathologic features of extrapulmonary EBM were assessed., Results: Fifty-six patients (33 females, 23 males; mean age 53 years) were identified with EBM. Diagnoses included lymphoma (21), breast adenocarcinoma (11), colonic adenocarcinoma (7), melanoma (6), renal cell carcinoma (RCC, 5), embryonal carcinoma (2), and 1 case each of tonsillar squamous cell carcinoma, thymic carcinoma, leiomyosarcoma, and sarcoma, not otherwise specified. The sensitivity of BB for identifying EBM was 85% overall and 94% for non-hematologic malignancies. The mean interval between primary diagnosis and EBM was 59 months (range 0-264 months). Excluding ten patients who had EBM at their initial presentation, lymphoma had the shortest (10 months) and RCC had the longest (264 months) mean interval between primary diagnosis and EBM. The mean time between EBM identification and death was 22.4 months (n = 24)., Conclusion: Bronchial brushing is a sensitive technique for diagnosing non-hematologic extrapulmonic endobronchial metastases.

Controlled invasion is essential during many physiological processes, whereas its deregulation is a hallmark of cancer. Here we demonstrate that embryonic, induced pluripotent and amniotic fluid stem cells share the property to induce the invasion of primary somatic cells of various origins through insulin-like growth factor I (IGF-I)- or II (IGF-II)-mediated paracrine activation of mechanistic target of rapamycin complex 1 (mTORC1). We propose a model in which downstream of mTORC1 this stem cell-induced invasion is mediated by hypoxia-inducible factor 1-alpha (HIF-1α)-regulated matrix metalloproteinases. Manipulating the IGF signalling pathway in the context of teratoma formation experiments demonstrates that human stem cells use this mechanism to induce invasion and thereby attract cells from the microenvironment in vivo. In this study we have identified a so far unknown feature of human stem cells, which might play a role for the development of stem cell-derived tumours.Cell invasion is required for several physiological processes but it is unknown if stem cells induce invasiveness in other cells. Here, the authors show that human stem cells secrete insulin-like growth factor, which in turn activates the mTORC1 pathway, initiating invasive behaviour and attracting other cells.

Previous studies have reported the miR-513b is located on the X chromosome and is preferentially expressed in testis. However, the underlying mechanisms of miR-513b involved in spermatogenesis remains unknown. In this study, we found that hsa-miR-513b-5p was highly expressed in the testes of infertile males with maturation arrest compared with normal controls. Overexpression of hsa-miR-513b-5p suppressed testicular embryonal carcinoma (NT2) cell proliferation and induced apoptosis in vitro, whereas silencing of hsa-miR-513b-5p reversed these effects. In addition, we found that interferon regulatory transcription factor 2 (IRF2) was a direct and functional target of hsa-miR-513b-5p. Silencing of endogenous IRF2 enhanced hsa-miR-513b-5p-mediated effects on cell proliferation in NT2 cells, whereas overexpression of IRF2 reversed these effects. Moreover, immunoblotting showed that overexpression of hsa-miR-513b-5p or silencing of endogenous IRF2 could promote the expression of P53. Moreover, overexpression of hsa-miR-513b-5p in the absence of p53 could also induce cell apoptosis. Together, our results suggest that hsa-miR-513b-5p suppresses NT2 cell proliferation and promotes P53 protein expression by targeting IRF2, and abnormal testicular hsa-miR-513b-5p expression may contribute to maturation arrest., (Copyright © 2017. Published by Elsevier B.V.)

So far, the understanding of germ cell cancer (GCC) pathogenesis is based on a model, where seminomas and non-seminomas represent distinct entities although originating from a common precursor termed germ cell neoplasia in situ (GCNIS). Embryonal carcinomas (ECs), the stem cell population of the non-seminomas, is pluri- to totipotent and able to differentiate into cells of all three germ layers, giving rise to teratomas or tumours mimicking extraembryonic tissues (yolk sac tumours, choriocarcinomas). With regard to gene expression, (epi)genetics and histology, seminomas are highly similar to GCNIS and primordial germ cells, but limited in development. It remains elusive, whether this block in differentiation is controlled by cell intrinsic mechanisms or by signals from the surrounding microenvironment. Here, we reviewed the recent literature emphasizing the plasticity of GCCs, especially of seminomas. We propose that this plasticity is controlled by the microenvironment, allowing seminomas to transit into an EC or mixed non-seminoma and vice versa. We discuss several mechanisms and routes of reprogramming that might be responsible for this change in the cell fate. We finally integrate this plasticity into a new model of GCC pathogenesis, allowing for an alternative view on the dynamics of GCC development and progression., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)