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Purpose: The aim of this study was to investigate the association between methionine (MET) metabolism and endocrine function of the pituitary gland in patients with suprasellar region tumor., Materials and Methods: Twenty patients with intracranial germinoma were included in this study. Initial staging and all surveillance MET PET/CT scans and comparable serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and thyroid stimulating hormone (TSH) were analyzed. The patients were divided into two groups according to tumor location, with tumors in the suprasellar region (condition) or not (control). MET uptake of the pituitary gland (i.e., SUVR [standardized uptake value ratio]) and levels of FSH, LH, TSH were compared in the condition and control groups and in the before and after treatment phases of each group., Results: The SUVR in the control group was like that found in normal pituitary glands in previous studies, whereas the SUVR of the untreated condition group was high and that of treated condition group was low with significance compared to the control group. Serum levels of pituitary hormones in before and after treatment condition groups were significantly lower than those in the control group. The FSH and LH levels of curatively treated patients in the control group were positively correlated with SUVR with respective ß values of 3.71 and 0.98 (p < .001). The TSH level of the treated condition group was negatively correlated with SUVR (ß = -1.02, p < .001)., Conclusion: This study is the first known investigation to examine the association between MET metabolism and endocrine function of the pituitary gland, and it confirmed that MET metabolism reflects endocrine function. A future study validating the result of correlation analysis is warranted., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Purpose: Reactive lymphocytes are substantial components of germinoma, which are believed to be related to the favorable prognosis of this intracranial tumor and better response to immunotherapy. However, the mechanisms managing the recruitment of lymphocytes are poorly understood. High endothelial venules (HEVs) are specialized blood vessels that play key roles in lymphocyte trafficking in Lymph nodes. These vessels are associated with lymphocyte infiltration in chronic inflammatory diseases and various malignant tumors, but their distribution and implications in germinoma are unknown. This study aimed to investigate the distribution and implications of HEVs in intracranial germinomas., Methods: We investigated the presence and distribution of HEVs in 42 germinomas by immunohistochemical staining of peripheral node addressin (PNAd) and transmission electron microscopic examination. The correlation of the densities of HEVs with the extent of T and B lymphocyte infiltration and several clinicopathological characteristics were also analyzed to determine whether HEVs are responsible for lymphocyte recruitment and their roles in anti-tumor immunity in germinoma., Results: PNAd-positive HEVs were detected in 31% (13/42) of germinomas, and their presence correlated with abundant infiltrating CD3+ T cells, CD20 + B cells and CD8+ cytotoxic T lymphocytes (p = 0.0410, 0.0023, and 0.0061, respectively). Higher HEVs density was also correlated with several clinicopathological parameters, which are recognized indicators for favorable prognosis in germinomas, including typical tumor location (p = 0.0093), lower tumor cell content (p = 0.0428), and younger age at diagnosis (p = 0.0121). Furthermore, bioinformatics analysis showed HEVs-associated genes mainly enriched in immune-related Gene Ontology terms, including innate immune response, inflammatory response, and B cell receptor signaling pathway. The xCell analysis revealed that germinomas with higher HEVs enrichment scores had increased levels of the immune score, microenvironment score, dendritic cells, CD8+ central memory T-cells, CD4+ memory T-cells, and B-cells., Conclusions: Our findings indicate that HEVs could contribute to lymphocyte recruitment in germinomas, thus may serve as a predictor of favorable prognosis and better response to immunotherapy in this intracranial tumor., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Objective: To investigate immunohistochemical patterns of CXorf67 and H3K27me3 proteins in central nervous system germ cell tumors (GCTs) and to assess their values in both diagnosis and differential diagnosis. Methods: A total of 370 cases of central nervous system GCTs were collected from 2013 to 2020 at Huashan Hospital of Fudan University, Shanghai, China. The expression of CXorf67, H3K27me3 and commonly-used GCT markers including OCT4, PLAP, CD117, D2-40, and CD30 by immunohistochemistry (EnVision method) was examined in different subtypes of central nervous system GCTs. The sensitivity and specificity of each marker were compared by contingency table and area under receiver operating characteristic (ROC) curve. Results: Of the 370 cases there were 282 males and 88 females with a mean age of 19 years and a median age of 17 years (range, 2-57 years). Among the GCTs with germinoma, the proportions of male patients and the patients with GCT located in sellar region were both higher than those of GCTs without germinoma ( P <0.05), respectively. CXorf67 was present in the nuclei of germinoma and normal germ cells, but not in other subtypes of GCT. H3K27me3 was negative in germinoma, but positive in the nuclei of surrounding normal cells and GCTs other than germinoma. In the 283 GCTs with germinoma components, the expression rate of CXorf67 was 90.5% (256/283), but no cases were positive for H3K27me3. There was also an inverse correlation between them ( r 2 =-0.831, P <0.01). The expression rates of PLAP, OCT4, CD117 and D2-40 were 81.2% (231/283), 89.4% (253/283), 73.9% (209/283) and 88.3% (250/283), respectively. In 63 mixed GCTs with germinoma components, the expression rate of CXorf67 was 84.1% (53/63), while all cases were negative for H3K27me3. The expression rates of PLAP, OCT4, CD117 and D2-40 were 79.4% (50/63), 79.4% (50/63), 66.7% (42/63) and 87.3% (55/63), respectively. The 6 markers with largest area under ROC curve in ranking order were H3K27me3, CXorf67, D2-40, OCT4, PLAP and CD117 ( P <0.05). Conclusions: CXorf67 and H3K27me3 have high sensitivity and high specificity in diagnosing germinoma. There is a significant inverse correlation between them. Therefore, they can both be used as new specific immunohistochemical markers for the diagnosis of GCTs.

Purpose: The purpose of this study was to investigate the value of C-11 methionine (MET) positron emission tomography (PET)/computed tomography (CT) in patients with intracranial germinoma (IG)., Material and Methods: We conducted a retrospective analysis of 21 consecutive patients with pathologically confirmed IGs and eight patients with intracranial non-germinomas (INGs) located in a similar region. Clinical characteristics, imaging findings, and tumor markers such as α-fetoprotein (AFP) and β-human chorionic gonadotropin (HCG) were used as clinical variables. Maximum standardized uptake value (SUVmax), tumor-to-normal tissue (T/N) ratio, and visual scoring of tumor were used as MET PET parameters., Results: All IGs were well visualized on MET PET with a three-grade visual scoring system. In addition, SUVmax of IGs was higher than that of INGs (P = 0.005). Pre-treatment (Pre-Tx) T/N ratio was significantly correlated with pre-Tx serum HCG (P = 0.031). Moreover, MET PET parameters showed significant associations with tumor location, sex, KRAS variant, and symptoms., Conclusion: MET PET/CT could be a useful diagnostic tool in patients suspected of having IGs. In addition, the MET avidity of tumor is a potential surrogate biomarker of HCG, which has been used as a diagnostic marker for IGs. Tumor MET parameters also had significant differences according to tumor locations, sex, symptoms, and KRAS mutation. However, MET avidity of tumors had no significant prognostic value., Competing Interests: The authors have declared that no competing interests exist.

Background: Germinomas (IG) account for up to 50% of all intracranial germ cell tumours. These tumours are reputed to be more prevalent in Oriental populations in comparison to Western cohorts. Biological characteristics of IG in other ethnic groups are unknown. Singapore is a multi-ethnic country with diverse cultures. Owing to inter-racial heterogeneity, the authors hypothesize there are molecular differences between paediatric IG patients in our local population. The aims of this study are exploratory: firstly, to identify molecular characteristics in this tumour type and circulating CSF unique to different racial cohorts; and next, to corroborate our findings with published literature., Methods: This is a single-institution, retrospective study of prospectively collected data. Inclusion criteria encompass all paediatric patients with histologically confirmed IG. Excess CSF and brain tumour tissues are collected for molecular analysis. Tumour tissues are subjected to a next generation sequencing (NGS) targeted panel for KIT and PDGRA. All CSF samples are profiled via a high-throughput miRNA multiplexed workflow. Results are then corroborated with existing literature and public databases., Results: In our cohort of 14 patients, there are KIT exon variants in the tumour tissues and CSF miRNAs corroborative with published studies. Separately, there are also KIT exon variants and miRNAs not previously highlighted in IG. A subgroup analysis demonstrates differential CSF miRNAs between Chinese and Malay IG patients., Conclusion: This is the first in-depth molecular study of a mixed ethnic population of paediatric IGs from a Southeast Asian cohort. Validation studies are required to assess the relevance of novel findings in our study.

Purpose: We performed a prospective single-arm trial (NCT02782754) to explore the feasibility of reducing radiation therapy (RT) dose when induction chemotherapy is combined in the treatment of intracranial germinoma with beta-human chorionic gonadotropin levels <200 mIU/mL., Methods and Materials: All patients aged 3 to 35 years from November 2012 to June 2018 were eligible for this study. Four cycles of induction chemotherapy were given before RT. Carboplatin/etoposide and cyclophosphamide/etoposide regimens were used in alternation every 3 weeks. A dose of 18 Gy of craniospinal RT for metastatic tumors, whole brain RT for basal ganglia tumors, or otherwise whole ventricular RT followed by 12.6 Gy of boost RT to the primary tumor bed was administered after induction chemotherapy. The primary endpoint of this study was progression-free survival., Results: A total of 41 consecutive patients were enrolled (location: suprasellar in 12, pineal in 12, both suprasellar and pineal in 11, and basal ganglia in 6 patients). Eleven patients had leptomeningeal seeding. Toxicity during chemotherapy was mild, except for bone marrow suppression. Tumor status after induction chemotherapy was complete response in 33 patients and partial response in 8. All but 2 patients completed the scheduled treatment. All patients but 1 remained event free during a median follow-up of 3.4 (range, 0.3-7.0) years from diagnosis. The 1 patient experienced relapse and died of tumor bleeding. Late effects were not significant except for neuroendocrine dysfunction already present at diagnosis. Vertical growth and cognitive function were not significantly disturbed by treatment., Conclusions: This study showed the feasibility of reducing RT dose/volume with induction chemotherapy in pathologically pure germinoma with elevated beta-human chorionic gonadotropin levels up to 200 mIU/mL., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aims: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown., Methods: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases., Results: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%)., Conclusions: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies., (© 2019 British Neuropathological Society.)

Intracranial germinoma sometimes present as bifocal germinoma, and whether bifocal germinoma should be treated as a synchronous or disseminated disease remains unclear. This study aimed to determine the optimal treatment modality for bifocal germinoma. Patients with bifocal germinoma who received radiotherapy (RT) from March 1990 to August 2017 were included for analysis. A total of 21 patients were included. The median follow-up period was 76.2 months (range, 6.2-305.4 months). There were 17 patients who received cranio-spinal irradiation (CSI) with local RT; 3, whole ventricular RT (WVRT) with local RT; and 1, local RT only. Three recurrences occurred (1 patient each among those who underwent CSI, WVRT, and local RT). Recurrence in the patient who received CSI and who received WVRT occurred in the right thalamus and right frontal convexity, respectively. Meanwhile, the patient who received local RT showed not only a recurred lesion in the hypothalamus, but also cerebrospinal fluid seeding. For this patient, salvage CSI was performed and complete response was achieved after treatment. However, after 9 years and 6 months, he was diagnosed with glioblastoma and expired. As for toxicity, although 17 patients showed decrease in complete blood count levels during treatment, all patients recovered soon after treatment completion. Our findings suggest that bifocal germinoma may be considered as a disseminated disease when considering the patterns of failure according to RT fields. In addition, patients who received CSI showed low acute toxicity rates. However, further studies are necessary to confirm these findings., Competing Interests: The authors have declared that no competing interests exist.

One histopathological characteristic of intracranial germinoma is abundant tumor-infiltrating lymphocytes (TILs) showing a two-cell pattern with large undifferentiated tumor cells. The programmed cell death 1 (PD-1)/programmed cell death 1 ligand (PD-L) axis has recently been recognized as an anti-tumor immune system. To evaluate intratumor immune status in intracranial germinoma, we examined expressions of PD-1 and PD-L1 (clone 28-8) and subtypes of TILs. Expressions of PD-1 and PD-L1 were detected immunohistochemically in 25 formalin-fixed, paraffin-embedded tumor specimens from 24 patients with intracranial germinoma consisting of 22 primary and 3 recurrent tumors. To evaluate subtypes of TILs, quantification of lymphocytes with CD3, CD8, CD4, and Foxp3 was performed. Statistical analyses were performed among PD-1, PD-L1 and subtypes of TILs. In 25 tumor tissue, expressions of PD-1 in TILs and PD-L1 in tumor cells were identified in 96% (24/25) and 92% (23/25), respectively. Expression of PD-1 was associated with CD3+ TIL density. Expression of PD-1 correlated with Foxp3+ TIL density and CD8+ TIL density, but not with CD4+ TIL density. Furthermore, expression of PD-1 correlated strongly with Foxp3+/CD4+ ratio. Taken together, increase of PD-1+ expression is associated with accumulation of Foxp3+ and CD8+ TILs. These findings intimate that PD-1/PD-L1 axis might shape the immune infiltration suggesting a modulation of the immune response and subsequent tumor growth in intracranial germinoma. Anti-PD-1 and anti-PD-L1 are potential immune therapeutic strategies in intracranial germinoma.

Intracranial pure germinomas in children generally respond well to standard chemo-radiotherapy. However, some patients are refractory to standard therapy and require additional treatment. To investigate the characteristics of this subgroup, we retrospectively analyzed the clinical features and treatment outcomes of a cohort of 21 patients with intracranial pure germinomas who were diagnosed between April 2002 and December 2016 at Ehime University Hospital in Japan. Pure germinoma diagnosis was verified by histological examination of the tumor after surgery, and all patients received standard chemo-radiotherapy. A suite of clinical features, including neuroimaging, human chorionic gonadotropin-β subunit (HCG-β), and α-fetoprotein (AFP) in the cerebrospinal fluid (CSF), as well as immunohistochemical expression of HCG-β, AFP, and Ki-67 in the tumor tissue were analyzed. Nineteen of the 21 patients had a complete response to standard chemo-radiotherapy without early recurrence of the tumors. Of these 19 patients, 17 did not have elevated CSF HCG-β levels or express HCG-β in the tumor tissue. However, the two patients who were refractory to standard therapy had elevated CSF HCG-β levels and expressed HCG-β in the tumor cells. These data suggest that patients with pure germinoma presenting with both an elevation of HCG-β in the CSF and HCG-β expression in the tumor tissue may be refractory to frontline treatment. These markers may predict aggressive germinoma and may ultimately facilitate the development of more effective treatment options.

Immunomodulation and tumor-induced tolerance is one of the central mechanisms in the oncogenesis of malignant and benign neoplasms. While numerous pathways have been described, signaling through the programmed death receptor 1 (PD-1) on T lymphocytes, via activation through its ligand, programmed death ligand 1 (PD-L1) expressed on tumor cells is one of the central pathways involved in tumor-induced tolerance. While the neoplastic component of germinomas of the CNS is the germ cell, these tumors also exhibit an abundance of quiescent tumor-infiltrating lymphocytes. We therefore investigated whether PD-L1 expression may be responsible for germinoma-induced T cell anergy, and if these tumors may be susceptible to immunotherapy. Pathologic specimens obtained from 21 cases of CNS germinomas between 2000 and 2016 were analyzed for the presence of PD-L1 and PD-1 expression by immunohistochemistry. Nineteen of 21 germinomas (90%) harbored germ cell components that stained positively for PD-L1. Positive lymphocyte staining for PD-L1 was evident in 16 cases. PD-1 expression was largely confined to lymphocytes; PD-L1 therefore may contribute to lymphocyte quiescence observed in these tumors. These results raise the possibility that immune checkpoint inhibitors such as nivolumab may have a therapeutic role in future treatment of germinomas.

Background and Objective: Pediatric basal ganglia germ cell tumors (GCTs) represent a rare subset of tumors about which little is known. We aimed to summarize the clinical features and radiological findings of this special subgroup of GCTs., Methods: From January 2010 to January 2015, 12 pediatric patients with basal ganglia GCTs were treated in our hospital. The clinical features, radiologic findings, diagnosis, treatment, and outcome of these patients were analyzed retrospectively. Our institutional diagnostic principle and treatment strategy of this disease were discussed., Results: GCTs accounted for 25.5% of all the pediatric basal ganglia tumors treated in our hospital. There were 9 male and 3 female patients with a mean age of 11.5 ± 2.1 years. The most common symptom was progressive hemiparesis (n = 9, 75%). The radiologic findings showed that the lesions predominately located in caput of caudate nucleus (n = 9, 75.0%), followed by lenticular nucleus (n = 3, 25.0%). Hemiatrophy was commonly observed (n = 8, 66.7%). Eight patients were diagnosed as having germinomas, and 4 patients as having nongerminomatous germ cell tumors. During the follow-up period, preoperative neurologic dysfunctions improved in 7 patients and remained stable in 3. Two patients developed new onset of neurologic dysfunction after the treatment. Two patients suffered from tumor recurrence., Conclusions: GCTs are not as rare as considered in pediatric basal ganglia tumors. They bear some distinctive clinical and radiologic features, which can help with the accurate diagnosis and successful management of such tumors., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Dr Robert E. Scully, who recognized and defined gonadoblastoma (GB), used the term "dissecting gonadoblastoma" (DGB) to describe variants with either an infiltrative type or diffuse pattern instead of the usual small nested arrangement. These patterns have not been emphasized in the literature. To investigate the features of DGB we examined 50 GBs microscopically and performed immunohistochemistry (IHC) in some. DGB was found in 38 (76%) GBs and was represented by 3 patterns. The most frequent was solid/expansile (n=26), consisting of large coalescent nests of germ cells, often (92%) interrupted by fibrovascular septa, with usually minor numbers of sex cord cells. Less frequent were small anastomosing nests (n=24) and cord-like arrangements (n=22) of germ cells irregularly distributed in a prominent stroma and with mostly inconspicuous sex cord cells. Most DGBs (24) showed >1 pattern and demonstrated the characteristic globular deposits of basement membrane, although these were often subtle. The germ cells in all patterns varied from spermatogonium-like to seminoma-like; OCT3/4 was positive only in the latter (7/7). The sex cord cells were small with dense, oval or angulated nuclei, inconspicuous nucleoli, and positivity for inhibin (9/9, strong), FOXL2 (9/9, strong), SF1 (8/9, strong), SOX9 (9/9, weak and focal), WT1 (5/7, variable), and calretinin (3/7, variable). Granulomas were present in 84% of germinoma foci, 13% of DGB foci, and 8% of classic GB foci. Twenty two of 38 DGBs had associated germinoma; 3 also had embryonal carcinoma, yolk sac tumor, and choriocarcinoma, respectively. Follow-up of 2 cases lacking an invasive tumor showed that both patients were disease free at 13 and 4.8 years after bilateral gonadectomy. We conclude that DGB is commonly seen with classic GB and displays identical IHC features, supporting it as a morphologic variant of GB. It appears likely that cord-like DGB is the earliest phase in a GB developmental continuum that may proceed successively into anastomosing, nested (classic GB), and solid/expansile patterns. DGB often mimics germinoma because of the large size of the nests, pseudoinfiltrative pattern of some cases, and inconspicuous sex cord cells. The presence of sex cord cells (identification aided by IHC for sex cord markers), the heterogenous morphology of the germ cells, and globules of basement membrane are useful differential features. The lack of a granulomatous reaction also favors DGB over germinoma. Mistaking DGB for GB with invasive germinoma may result in more aggressive therapy than warranted. The likely relationship of DGB to the relatively recently described concept of so-called "undifferentiated gonadal tissue" is discussed herein.

CNS germinomas represent a unique germ cell tumor entity characterized by undifferentiated tumor cells and a high response rate to current treatment protocols. Limited information is available on their underlying genomic, epigenetic and biological alterations. We performed a genome-wide analysis of genomic copy number alterations in 49 CNS germinomas by molecular inversion profiling. In addition, CpG dinucleotide methylation was studied by immunohistochemistry for methylated cytosine residues. Mutational analysis was performed by resequencing of candidate genes including KIT and RAS family members. Ras/Erk and Akt pathway activation was analyzed by immunostaining with antibodies against phospho-Erk, phosho-Akt, phospho-mTOR and phospho-S6. All germinomas coexpressed Oct4 and Kit but showed an extensive global DNA demethylation compared to other tumors and normal tissues. Molecular inversion profiling showed predominant genomic instability in all tumors with a high frequency of regional gains and losses including high level gene amplifications. Activating mutations of KIT exons 11, 13, and 17 as well as a case with genomic KIT amplification and activating mutations or amplifications of RAS gene family members including KRAS, NRAS and RRAS2 indicated mutational activation of crucial signaling pathways. Co-activation of Ras/Erk and Akt pathways was present in 83% of germinomas. These data suggest that CNS germinoma cells display a demethylated nuclear DNA similar to primordial germ cells in early development. This finding has a striking coincidence with extensive genomic instability. In addition, mutational activation of Kit-, Ras/Raf/Erk- and Akt- pathways indicate the biological importance of these pathways and their components as potential targets for therapy.

Metastatic intracranial germinoma is difficult to treat. Although the proto-oncogene KIT is recognized as one of the most frequent genetic abnormalities in CNS germinoma, the development of new target therapeutic agents for CNS germinoma is hampered by the lack of clinically-relevant animal models that replicate the mutated or over-expressed KIT. CNS germinoma tumor cells from five pediatric patients were directly implanted into the brains of Rag2/severe combined immune deficiency mice. Once established, the xenograft tumors were sub-transplanted in vivo in mouse brains. Characterization of xenograft tumors were performed through histologic and immunohistochemical staining, and KIT mutation analysed with quantitative pyro-sequencing. Expression of putative cancer stem cell markers (CD133, CD15, CD24, CD44, CD49f) was analyzed through flow cytometry. Two patient-derived orthotopic xenograft (PDOX) models (IC-6999GCT and IC-9302GCT) were established from metastatic germinoma and serially sub-transplanted five times in mouse brains. Similar to the original patient tumors, they both exhibited faint expression (+) of PLAP, no expression (-) of β-HCG and strong (+++) expression of KIT. KIT mutation (D816H), however, was only found in IC-9320GCT. This mutation was maintained during the five in vivo tumor passages with an increased mutant allele frequency compared to the patient tumor. Expression of putative cancer stem cell markers CD49f and CD15 was also detected in a small population of tumor cells in both models. This new pair of PDOX models replicated the key biological features of pediatric intracranial germinoma and should facilitate the biological and pre-clinical studies for metastatic intracranial germinomas.

Testicular germ cell cancer develops from premalignant intratubular germ cell neoplasia, unclassified cells that are believed to arise from failure of normal maturation of fetal germ cells from gonocytes (OCT4(+)/MAGEA4(-)) into pre-spermatogonia (OCT4(-)/MAGEA4(+)). Intratubular germ cell neoplasia cell subpopulations based on stage of germ cell differentiation have been described, however the importance of these subpopulations in terms of invasive potential has not been reported. We hypothesized that cells expressing an immature (OCT4(+)/MAGEA4(-)) germ cell profile would exhibit an increased proliferation rate compared with those with a mature profile (OCT4(+)/MAGEA4(+)). Therefore, we performed triple immunofluorescence and stereology to quantify the different intratubular germ cell neoplasia cell subpopulations, based on expression of germ cell (OCT4, PLAP, AP2γ, MAGEA4, VASA) and proliferation (Ki67) markers, in testis sections from patients with preinvasive disease, seminoma, and non-seminoma. We compared these subpopulations with normal human fetal testis and with seminoma cells. Heterogeneity of protein expression was demonstrated in intratubular germ cell neoplasia cells with respect to gonocyte and spermatogonial markers. It included an embryonic/fetal germ cell subpopulation lacking expression of the definitive intratubular germ cell neoplasia marker OCT4, that did not correspond to a physiological (fetal) germ cell subpopulation. OCT4(+)/MAGEA4(-) cells showed a significantly increased rate of proliferation compared with the OCT4(+)/MAGEA4(+) population (12.8 versus 3.4%, P<0.0001) irrespective of histological tumor type, reflected in the predominance of OCT4(+)/MAGEA4(-) cells in the invasive tumor component. Surprisingly, OCT4(+)/MAGEA4(-) cells in patients with preinvasive disease showed significantly higher proliferation compared to those with seminoma or non-seminoma (18.1 versus 10.2 versus 7.2%, P<0.05, respectively). In conclusion, this study has demonstrated that OCT4(+)/MAGEA4(-) cells are the most frequent and most proliferative cell population in tubules containing intratubular germ cell neoplasia, which appears to be an important factor in determining invasive potential of intratubular germ cell neoplasia to seminomas.

In this study, 10 patients with biopsy-proven germinoma with a beta-human chorionic gonadotropin (β-HCG) level >50 mIU/ml received intensive chemotherapy followed by reduced-dose radiotherapy (RT) to reduce late effects from RT. CSF β-HCG levels were >200 mIU/ml in five patients. After endoscopic or stereotactic biopsy, four cycles of induction chemotherapy were administered prior to RT. A CEB regimen (carboplatin + etoposide + bleomycin) and a CyEB regimen (cyclophosphamide + etoposide + bleomycin) were alternated. No residual tumor remained after induction chemotherapy in six patients, only cystic lesions were present at the primary tumor site in three, and a small solid residual tumor was observed in the remaining patient; however, all these patients had normal β-HCG levels. If complete response was achieved before initiation of RT, 19.5 Gy craniospinal RT (CSRT) + 10.8 Gy local RT was administered to the tumor bed. If residual lesion was suspected, the dose of RT was selected according to the presence/absence of tumor dissemination at diagnosis (19.5 Gy CSRT + 19.8 Gy local RT for localized tumors and 24.0 Gy CSRT + 16.2 Gy local RT for disseminated tumors). Eight patients, including four patients with a β-HCG level >200 mIU/ml, received 19.5 Gy CSRT. All patients remain disease free at a median follow-up of 58 (range 35-94) months from diagnosis. Our data suggest that pathologically pure germinoma with a significantly elevated β-HCG level might be cured with reduced-dose RT if intensive chemotherapy is provided.

The transcription factor OCT4 is an established diagnostic marker for central nervous system (CNS) germinoma. However, no data are available to date concerning the expression of its downstream target undifferentiated embryonic cell transcription factor 1 (UTF1) in CNS germ cell tumours. We examined 21 CNS germinomas and two mixed CNS germ cell tumours for UTF1 and the post-transcriptional regulator LIN28 immunohistochemical expression. We compared the profile to established diagnostic germinoma markers and to the expression in six testicular and four metastatic germ cell tumours as well as 150 CNS tumours of various backgrounds. We found UTF1 expression in 23 of 23 and LIN28 in 20 of 23 CNS germ cell tumours. The established germinoma markers cKIT (23/23), OCT4 (21/23) and placental alkaline phosphatase (PLAP) (19/21) were also frequently expressed in our cohort. In terms of signal intensity and frequency, UTF1 showed similar results as cKIT but staining was superior to OCT4, PLAP and LIN28. OCT4 was absent in all CNS metastases and haemangioblastomas, while UTF1 was weakly observed in two metastases.With a sensitivity of 100% and a specificity of 97% in the detection of CNS germinomas, UTF1 serves as a new reliable alternative in the diagnostic setting of CNS germ cell tumours.

Intracranial germ cell tumors (iGCTs) are the second most common brain tumors among children under 15 in Japan. The pathogenesis of iGCTs is largely unexplored. Although a subset of iGCTs is known to have KIT mutation, its impact on the biology and patients' survival has not been established. In this study, we investigated genes involved in the KIT signaling pathway. 65 iGCTs (30 pure germinomas, 14 teratomas, 18 mixed GCTs, 2 yolk sac tumors, 1 choriocarcinoma) were screened for mutation of KIT, KRAS, NRAS, HRAS, BRAF, PDGFRA, and IDH1 by direct sequencing. KIT expression was examined by immunohistochemistry and quantitative PCR. Chromosomal status was analyzed by array-comparative genomic hybridization (aCGH). Somatic mutations were detected only in KIT and RAS, which were frequently observed in pure germinomas (60.0 %), but rare in non-germinomatous GCTs (NGGCTs) (8.6 %). All KIT/RAS mutations were mutually exclusive. Regardless of the mutation status or mRNA expression, the KIT protein was expressed in all germinomas, while only in 54.3 % of NGGCTs. Amplification of KIT was found in one pure germinoma by aCGH. In pure germinomas, high expression of KIT mRNA was associated with the presence of KIT/RAS alterations and severe chromosomal instability. Our results indicate that alterations of the KIT signaling pathway play an important role in the development of germinomas. Pure germinomas may develop through two distinct pathogeneses: one with KIT/RAS alterations, elevated KIT mRNA expression and severe chromosomal instability, and the other through yet an unidentified mechanism without any of the above abnormalities.

Extragonadal germ cell tumors rarely arise from the brain stem. In this report, the authors present a rare case of germinoma developing in the medulla oblongata. A 31-year-old woman complained of intractable hiccups, hoarseness, and swallowing disturbance. Magnetic resonance imaging showed a tumor located on the dorsal part of the medulla oblongata without any abnormalities in the pineal body and suprasellar regions. Histological diagnosis of surgically resected tumor was germinoma. Tumor cells were positive for alkaline phosphatase, c-kit, octamer-binding transcription factor 3/4, cytokeratin, and epithelial membrane antigen in immunohistochemistry. The patient received adjuvant chemotherapy and irradiation to the residual tumor and cerebral ventricles. The patient is alive without recurrence 6 months after the surgery.

OCT4 expresses variably in primary intracranial germinomas. In this study, we tested our hypothesis that such variation of OCT4 is predictive of outcome in primary intracranial germinomas. Thirty-one histologically identified CNS germinoma patients were enrolled in our study. We collected medical data, immunohistochemically evaluated the OCT4 expression level, and followed up all patients from April 2001 to May 2010. We found that 7 of the 31 patients expressed OCT4 weakly, 11 expressed OCT4 moderately, and 13 expressed OCT4 strongly. No significant correlation between the OCT4 expression level and clinicopathological characteristics was observed. WV-CS combined treatment modality showed a better 5-year progression-free survival (PFS) rate than other treatment modalities and a low expression level of OCT4 showed a significantly better 5-year PFS. In both the WV-CS combined treatment modality and other treatments modality group, patients received a better 5-year PFS and had a lower level of OCT4 expression. As a result, we suggest OCT4 as a probable prognostic marker for intracranial germinoma.

Six of 276 prepubertal testicular specimens had atypical germ cells. Specimens from 3 intersex patients met the criteria for intratubular germ cell neoplasia, unclassified type (IGCNU). Of the remaining 3 specimens (2 patients), 1 from a Down syndrome patient had centrally positioned germ cells with mild nuclear enlargement and hyperchromasia and expressed fetal germ cell markers (Oct3/4, placental alkaline phosphatase, CD117); it was classified as "delayed germ cell maturation". The remaining 2 specimens (1 patient) were diagnosed as IGCNU but, in retrospect, the patient had an undiagnosed intersex disorder. We therefore did not find a bona fide case of IGCNU in prepubertal testes apart from an intersex disorder.

The novel stem cell-related gene, HESRG, was first identified by our group, and its expression pattern in human tumors remains unknown. In this study, we used RT-PCR to systematically investigate the expression of HESRG in various types of intracranial tumors and found that HESRG was expressed only in germinoma and embryonal carcinoma, but hardly at all in other types of brain tumors. Real-time PCR results further confirmed this expression pattern. Subsequently, we tested 134 intracranial non-germ cell tumors and 64 intracranial germ cell tumors by immunohistochemistry. Our results showed that HESRG was expressed strongly and diffusively in the nuclei of tumor cells in intracranial germinoma and embryonal carcinoma as well as in human embryonic stem cells. No positive staining signal was observed in any other type of intracranial tumors. In germinomas, 25 of 31 showed intensive (3+) expression, four cases showed moderate (2+) immunostaining and the remaining 2 cases showed weak (1+) immunostaining. In embryonal carcinoma, 6 of 9 showed intensive (3+) immunostaining and 3 of 9 showed moderate (2+) immunostaining. These results suggest that HESRG is a novel, sensitive and specific biomarker for intracranial germinoma and embryonal carcinoma.

Intracranial germ cell tumors constitute a unique group of tumors, more often reported from the Asian region. Amongst them, the non-germinomatous variety occurs with a lesser frequency than the germinomatous variety. We report two children with mixed germ cell tumors with unusual clinical presentations: Central diabetes insipidus and recent-onset oculomotor palsy mimicking pituitary apoplexy. Unlike in adults, suprasellar lesions with a pituitary apoplexy-like picture in the pediatric age group may suggest a possibility of a mixed germ cell tumor.

Intracranial germ cell tumors (GCTs) typically affect children and adolescents. We here report on a 59-year-old male patient presenting with diplopia, polydipsia and polyuria. On clinical examination, slight restriction of the upward gaze was seen on the left side. Computed tomography demonstrated calcifications in the pineal region and enhanced neurohypophysis. Magnetic resonance imaging displayed a heterogeneous pineal mass of 3-cm diameter, which was multicystic with an enhanced cyst wall, and also swelling of the pituitary stalk. The pineal lesion of the tumor, which included calcifications and keratinaceous components, was totally excised using an occipital transtentorial approach. Histopathological examination showed it to be a mixed GCT with germinoma and mature teratoma components. Postoperative chemoradiotherapy provided complete disappearance of the suprasellar lesion. To our knowledge, this is the first case of mixed bifocal GCT in an older adult reported in the literature, although a few cases of tumors with a single histological component have been reported. Hence, our case further underlines the possibility of the occurrence of GCTs in older adults and advocates the consideration of GCTs in the differential diagnosis of such cases for appropriate management.

Central nervous system tumors are the second most common type of childhood cancer in Turkey. Germinomas constitute two thirds of intracranial germ cell tumors. The granulomatous inflammation occurring around germinomas can cause histological diagnostic difficulty. We present a 12-year-old girl with a diagnosis of germinoma in the corpus callosum associated with granulomatous reaction to emphasize the diagnostic challenge which may occur during stereotactic biopsy interpretation or intraoperative consultation.

Aquaporins (AQP) are water-channel proteins with roles in tumour cell migration, angiogenesis, cerebral oedema and cell-cell adhesion. We aimed to determine the expression of AQP1 and AQP4 in paediatric brain tumours. Twenty tumour bank specimens were subject to Western blot analysis and quantitative polymerase chain reaction (PCR) to determine the expression of AQP1 and 4. Immunohistochemical staining was used to determine the distribution of AQP1 and 4 expression. Medulloblastomas, primitive neuroectodermal tumour, germinomas and higher grade gliomas did not express AQP1 and 4. Of the ependymomas, those in the posterior fossa all demonstrated markedly increased expression of AQP1 and 4. A supratentorial ependymoma demonstrated a moderate increase in AQP1 but not AQP4. Pilocytic astrocytomas demonstrated high levels of AQP1 and 4 but had a more variable pattern of staining. AQP1 and 4 have relevance to paediatric brain tumours and are worthy of further investigation in developing potential therapeutic strategies., (Elsevier Ltd. All rights reserved.)

Pathologic stage and postsurgical treatment guidelines of malignant germ cell tumors, currently take into account angiolymphatic invasion, degree of extra testicular invasion, and serum tumor marker levels. The significance of rete testis invasion by malignant germ cell tumors or intratubular germ cell neoplasia however remains controversial. A search through the surgical pathology and expert consultation files at our institution from 2002 to 2009 was made for malignant germ cell tumors and intratubular germ cell neoplasia in orchiectomy specimens. Clinicopathologic data including rete testis status were obtained. Two hundred ninety-two orchiectomy specimens were identified. One hundred thirty-six were associated with malignant germ cell tumors. Mean patient age was 33 years (range, 14-67 years). The mean greatest tumor dimension was 4.1 cm (range, 0.8-18 cm). Fifty-six were pure seminoma (40%), 50 were nonseminomatous malignant germ cell tumors (35%), and 35 were mixed malignant germ cell tumors including a seminoma component (25%). Intratubular germ cell neoplasia was identified in 99 cases (70%). Pathologic stage at presentation was as follows: stage 1, 71 patients (50%); stage 2, 62 patients (45%); stage 3, 2 patients (1%); and indeterminate, 6 patients (4%). Seventy-eight patients had documented rete testis status: rete testis invasion, 41 (53%); no rete testis invasion, 37 (47%). Angiolymphatic invasion was present in 62 cases (44%). Follow-up information was available in 43 patients with known rete testis status. Mean follow-up duration was 43 months (range, 3-65 months). Twenty patients had rete testis invasion, and 23 patients had no rete testis invasion. Intratubular germ cell neoplasia was present in patients with rete testis invasion in 18 cases (90%), compared to only 13 cases (57%) in patients without rete testis invasion, P = .02. Serum markers were elevated in 10 patients (50%) with rete testis invasion compared to only 6 patients (26%) without rete testis invasion, P = .05. The combination of rete testis invasion and angiolymphatic invasion were present in 8 cases and were found to be associated with elevated serum tumor markers in 7 (88%) of the 8 cases, compared to the combination of no invasion of the rete testis and angiolymphatic invasion showing elevated serum tumor markers in 3 (38%) of 8 cases. However, 7 patients (35%) with rete testis invasion developed metastatic disease, and 11 patients (48%) without rete testis invasion developed metastatic disease. Rete testis status should be documented in orchiectomy specimens with malignant germ cell tumors. Intratubular germ cell neoplasia may be the only component of a malignant germ cell tumor involving the rete testis. In this series, elevated tumor markers were more likely associated with angiolymphatic invasion and positive rete testis status. Positive rete testis status does not appear to be an independent predictor of patient outcome., (Copyright 2010 Elsevier Inc. All rights reserved.)

Purpose: It has been documented that testicular germ cell cancer (TGCC) patients may be at increased risk of developing emotional distress (EMD). Hence, the aim of the present study was to investigate whether EMD is related to the presence of hypogonadism, androgen receptor (AR) polymorphism and/or treatment intensity., Patients and Methods: Three to five years after treatment, testosterone and luteinizing hormone (LH) levels were measured in 165 TGCC patients. These patients also completed a questionnaire concerning mental health. EMD was measured by the Hospital Anxiety and Depression Scale (HADS). The androgen receptor (AR) gene has two polymorphic regions in exon I; glutamine encoding CAG and glycine encoding GGN repeats. Association between emotional disorders and AR polymorphisms as well as type of treatment was assessed., Results: Neither anxiety (OR 1.0; 95% CI 0.40-2.4) nor depression (OR 1.1; 95% CI 0.20-6.4) were overrepresented in biochemically hypogonadal TGCC patients and no association between AR polymorphisms and EMD was found. Patients treated with >or=5 cycles of cisplatinum based chemotherapy due to refractory or relapsed disease were more prone to experiencing symptoms of anxiety (p=0.006), but not depression (p=0.38)., Conclusions: Biochemical hypogonadism and AR polymorphism do not seem to be risk factors for EMD in TGCC patients. Patients with refractory or relapsed disease receiving >or=5 cycles of cisplatinum based chemotherapy may, to a higher degree than patients receiving less intense therapy, suffer from anxiety., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Germ cell tumors are a heterogeneous group of neoplasms derived from residual primordial tissue. These tumors are commonly found in the brain, testes, or ovaries, where they are termed germinomas, seminomas, or dysgerminomas, respectively. Like several other tumor types, germ cell tumors often harbor an immune cell infiltrate that can include substantial numbers of B cells. Yet little is known about whether the humoral immune response affects germ cell tumor biology. To gain a deeper understanding of the role B cells play in this tumor family, we characterized the immune cell infiltrate of all three germ cell tumor subtypes and defined the molecular characteristics of the B cell Ag receptor expressed by tumor-associated B cells. Immunohistochemistry revealed a prominent B cell infiltrate in the microenvironment of all tumors examined and clear evidence of extranodal lymphoid follicles with germinal center-like architecture in a subset of specimens. Molecular characterization of the Ig variable region from 320 sequences expressed by germ cell tumor-infiltrating B cells revealed clear evidence of Ag experience, in that the cardinal features of an Ag-driven B cell response were present: significant somatic mutation, isotype switching, and codon insertion/deletion. This characterization also revealed the presence of both B cell clonal expansion and variation, suggesting that local B cell maturation most likely occurs within the tumor microenvironment. In contrast, sequences from control tissues and peripheral blood displayed none of these characteristics. Collectively, these data strongly suggest that an adaptive and specific humoral immune response is occurring within the tumor microenvironment.

Combined action of SOX and POU families of transcription factors plays major roles in embryonic development. In embryonic stem cells, the combination of SOX2 and POU5F1 (OCT3/4) is essential for maintaining the undifferentiated state by activating pluripotency-linked genes, and inhibition of genes involved in differentiation. Besides embryonic stem cells, POU5F1 is also present in early germ cells, primordial germ cells, and gonocytes, where it has a role in suppression of apoptosis. Here we demonstrate that SOX2 is absent in germ cells of human fetal gonads, and as expected carcinoma in situ (CIS), ie the precursor lesion of testicular germ cell tumours of adolescents and adults (TGCTs), and seminoma. Based on genome-wide expression profiling, SOX17 was found to be present, instead of SOX2, in early germ cells and their malignant counterparts, CIS and seminoma. Immunohistochemistry, western blot analysis, and quantitative RT-PCR showed that SOX17 is a suitable marker to distinguish seminoma from embryonal carcinoma, confirmed in representative cell lines. Aberrant SOX2 expression can be present in Sertoli cells when associated with CIS, which can be misdiagnosed as embryonal carcinoma. In conclusion, this study demonstrates the absence of SOX2 in human embryonic and malignant germ cells, which express SOX17 in conjunction with POU5F1. This finding has both diagnostic and developmental biological implications. It allows the identification of seminoma-like cells from embryonal carcinoma based on a positive marker and might be the explanation for the different function of POU5F1 in normal and malignant germ cells versus embryonic stem cells.

Central nervous system (CNS) germ cell tumors constitute a unique class of rare tumors that mainly affect children and adolescents. These tumors are believed to originate from displaced primordial germ cells. Recently, results of treatment of germ cell tumors have improved with use of radiotherapy and combination chemotherapy. However, some tumors have proven refractory to intensive treatment with surgery, radiation, and combination chemotherapy. Nestin is an intermediate filament protein expressed in undifferentiated cells during CNS development and in CNS tumors and is used as a marker of immature elements of tumors, including brain tumor stem cells. In this study, we examined for the first time nestin expression in 19 CNS germ cell tumors (nine pure germinomas, five germinomas with syncytiotrophoblastic giant cells, one yolk sac tumor, one choriocarcinoma, one embryonal carcinoma, and two mature teratomas). Nestin was expressed in 14 cases but was not expressed in three pure germinomas and two mature teratomas. Clinically, nestin-negative tumors did not exhibit dissemination, while all tumors that exhibited dissemination also strongly expressed nestin protein. These findings suggest that the detection of nestin expression could be useful in the management of CNS germ cell tumors, as an auxiliary predictor of dissemination and/or progression.

Objective: To study the clinicopathologic features, immunohistochemical findings and prognosis of primary diffuse large B-cell lymphoma (DLBCL) of testis., Methods: Fourteen cases of primary DLBCL of testis, diagnosed according to the 2001 World Health Organization staging standards for hematopoietic and lymphoid tumors, were retrospectively studied. Immunohistochemical study was performed and follow-up information analyzed., Results: The median age of the patients was 62 years. There were 10 patients in stage I, 3 in stage II and 1 in stage IV. Follow-up information was available in 11 patients (78.6%). Three of which were still alive and eight died (with duration of survival ranging from 5 to 19 months). The patients usually presented with unilateral painless enlargement of testis. Histologically, the lymphoma cells of all cases showed a centroblastic appearance. One case belonged to the germinal center B cell-like subtype on immunohistochemical study, while the remaining 13 cases were classified as non-germinal center B cell-like subtype. Ten of the 14 cases (71.4%) showed overexpression of p53 protein. Most cases demonstrated high proliferation index. Six of the 14 cases (42.9%) were positive for bcl-2 protein. The overall 1-year, 2-year and 5-year survival rates were 45.5%, 17.0% and 17.0%, respectively., Conclusions: Most cases with primary DLBCL of testis were of peripheral activated B-cell origin. The prognosis is usually not favorable, with propensity of local relapse and systemic dissemination. Accurate pathologic diagnosis relies on detailed histologic examination and immunohistochemical study.

We report a case of a 24-year-old man with a right thalamic germinoma that initially mimicked a granulomatous inflammation, compatible with neurosarcoidosis based on clinical symptoms, imaging results and histology of an endoscopically navigated biopsy. A second biopsy, prompted by clinical course, and performed openly from parieto-lateral revealed the underlying germinoma, obscured in the first biopsy by a granulomatous tissue response, particularly at the tumor edge. The present case highlights granulomatous inflammatory tissue response on the tumor edge of germinoma as a tumor-immanent diagnostic challenge. This diagnostic problem is aggravated by stereotactic and endoscopic approaches. We conclude that granulomatous inflammation in a specimen obtained by biopsy of a midline lesion should always be considered for the differential diagnosis of germinoma. Stereotactic and endoscopic surgery should sample several different target points within the lesion. Because of tumor heterogeneity of germinoma, the open biopsy approach is advantageous compared to endoscopic or stereotactic techniques for germinoma and should be considered if a germinoma is in the differential diagnosis and if allowed by the clinical situation.

Context: Immunohistochemistry (IHC) has become an important tool in the diagnosis of brain tumors., Objective: To review the latest advances in IHC in the diagnostic neuro-oncologic pathology., Data Sources: Original research and review articles and the authors' personal experiences., Data Synthesis: We review the features of new, useful or potentially applicable marker antibodies as well as the new uses of already established antibodies in the area of diagnostic neuro-oncologic pathology, focusing on the use of IHC for differential diagnosis and prognosis. We discuss (1) placental alkaline phosphatase, c-Kit, and OCT4 for germinoma, (2) alpha-inhibin and D2-40 for capillary hemangioblastoma, (3) phosphohistone-H3 (PHH3), MIB-1/Ki-67, and claudin-1 for meningioma, (4) PHH3, MIB-1/Ki-67, and p53 for astrocytoma, (5) synaptophysin, microtubule-associated protein 2, neurofilament protein, and neuronal nuclei for medulloblastoma, (6) INI1 for atypical teratoid/rhabdoid tumor, and (7) epithelial membrane antigen for ependymoma. All the markers presented here are used mainly for supporting or confirming the diagnosis, with the exception of the proliferation markers (MIB-1/Ki-67 and PHH3), which are primarily used to support grading and are reportedly associated with prognosis in certain categories of brain tumors., Conclusions: Although conventional hematoxylin-eosin staining is the mainstay for pathologic diagnosis, IHC has played a major role in differential diagnosis and in improving diagnostic accuracy not only in general surgical pathology but also in neuro-oncologic pathology. The judicious use of a panel of selected immunostains is unquestionably helpful in diagnostically challenging cases. In addition, IHC is also of great help in predicting the prognosis for certain brain tumors.

The homeodomain transcription factor, NANOG, along with OCT3/4 (POU5F1) and SOX2, is part of the core set of transcription factors that maintain embryonic stem cell self-renewal and pluripotency. Expression of NANOG has been detected in fetal germ cells and in gonadal germ cell tumors. To assess the diagnostic utility of NANOG in central nervous system (CNS) germ cell tumors, we analyzed its expression by immunohistochemistry in a series of 12 CNS germinomas and compared its expression with other stem cell markers. Strong nuclear expression of NANOG was demonstrated in >90% of the tumor cells in all cases. In contrast, expression of OCT3/4 and placental alkaline phosphatase was inconsistent and SOX2 was expressed in only rare cells. NANOG was not detected in tumor types frequently considered in the differential diagnosis of CNS germinoma: pineoblastoma, primitive neuroectodermal tumors, medulloblastoma, lymphoma, pituitary adenoma, atypical teratoid/rhabdoid tumor, Langerhans cell histiocytosis, and gliomas. These findings demonstrate that NANOG is a sensitive and specific marker of CNS germinoma. Compared with other currently used markers, NANOG may have superior diagnostic characteristics and can facilitate identification of germinomas in minute surgical biopsies commonly obtained from these tumors. These findings also suggest a potential biologic role for NANOG in maintenance of CNS germinoma.

Objective: Hyperglycosylated human chorionic gonadotropin (hCG-H) is a carbohydrate variant of hCG with double-sized oligosaccharide side chains. While hCG-H is produced exclusively by stem cytotrophoblast cells in gestational choriocarcinoma, by pregnancy cytotrophoblast at implantation and by the cytotrophoblast produced in testicular malignancies, regular hCG is produced only by differentiated syncytiotrophoblast cells., Study Design: hCG-H was measured using the Nichols Advantage hCG-H assay (Nichols Institute Diagnostics, San Clemente, California)., Results: hCG-H has a function separate from regular hCG. hCG-H, but not regular hCG, acts in vivo and in vitro to promote invasion, whether invasion through membranes or tumor formation. Invasion or tumorigenesis is completely blocked by administration of specific antibody to hCG-H. The same hCG-H-modulated invasion mechanisms are observed in early pregnancy, gestational choriocarcinoma and testicular cancers., Conclusion: hCG-H is a cytokinelike molecule, produced by cells different from those that make regular hCG and having a completely separate function. It appears to be the modulator of invasion as in implantation of pregnancy, gestational choriocarcinoma and testicular cancer malignancy.

Aims: To compare the suitability of new seminoma markers including transcription factors AP-2gamma, OCT3/4 and M2A for detection of metastatic and extragonadal seminomas with the two well-known markers c-KIT and PLAP., Materials and Methods: The immunohistochemical distribution of PLAP, c-KIT, M2A, AP-2gamma and OCT3/4 was examined in two pineal germinomas, 28 metastatic seminomas and 10 of their testicular primaries. Evaluation of specificity was achieved by additional tissue array studies on 75 malignancies other than germ cell tumours (GCT). Clinical data including serum PLAP were available in 18 patients., Results: Compared with other markers, significantly better staining results were observed with antibodies to M2A and AP-2gamma in all seminomatous GCT. In contrast, the staining pattern with antibodies to c-KIT, PLAP and OCT3/4 was variable or absent. The lowest specificity was obtained with c-KIT, which was expressed in a variety of non-GCT. The only M2A+ mesothelioma expressed no other seminoma markers. No correlation between serum PLAP level and tissue PLAP expression was found., Conclusions: M2A and AP-2gamma are the most sensitive markers for seminoma metastases or primary extragonadal seminomas. Combination of these markers provides highly specific and clear results for detection of a seminomatous GCT.

An 8-year-old boy presented with a rare case of germinoma involving the bilateral basal ganglia and cerebral white matter manifesting as precocious puberty. Magnetic resonance (MR) imaging at the initial presentation demonstrated mild hyperintense areas in the bilateral basal ganglia and corpus callosum on T1-weighted images, and a small hyperintense spot in the right internal capsule on T2-weighted images. Human chorionic gonadotropin (hCG) level was elevated in the cerebrospinal fluid (CSF), so we strongly suspected that threre was a hCG-producing germinoma originating in the bilateral basal ganglia. Stereotactic biopsy was performed. Histological examination revealed two-cell pattern germinoma. After three cycles of combination chemotherapy consisting of ifosfamide, cisplatin, and etoposide, followed by whole brain irradiation with a total dose of 24 Gy, the CSF hCG level fell below the detection limit, but MR imaging demonstrated no significant change. Intracranial hCG-producing germinoma should be suspected in patients presenting with precocious puberty and elevated CSF hCG level. Moreover, slight intensity change on MR imaging is important to identify germinoma arising from the basal ganglia in the early stage.

The risk of subsequent development of testicular germ cell neoplasia is related to presence of underlying developmental defects such as cryptorchidism, in which the risk is around 0.5%, and XY intersex with abdominal testes, in which the risk may be as high as 20-25%. We examined the hypothesis that the increased risk of germ cell malignancy in intersex testes with Y chromosome was a direct consequence of an abnormal increase in number of PLAP/CD117+ immature germ cells into postnatal life. Archival cases of uncomplicated cryptorchidism (CO) and XY intersex (INT) were identified and anonymized, and a subgroup of aged-matched cases had sections immunostained with placental alkaline phosphatase (PLAP) and CD117. From a total of 89 intersex and 105 cryptorchid cases identified, a power calculation to detect a 20% difference in expression between groups (alpha = 0.05, power = 80%) determined that 18 intersex and 36 cryptorchid cases were required. Thus, 58 cases were examined, median age 3 (range birth-11) years, including 39 CO and 19 INT. The prevalence of any PLAP+ germ cells was 2/39 (5.1%) versus 3/19 (15.7%), respectively. (Z = 1.4, p = 0.17). In contrast, 94% of cases showed presence of any CD117+ germ cells, but the frequency of CD117+ cells was not significantly different between groups (t = 0.56, p = 0.58). CD117 and PLAP identify different populations of germ cells in pediatric testes. The extent of increased risk of malignancy in XY INT is not simply related to increased numbers of immature PLAP+/CD117+ germ cells present; additional factors play a pathogenic role.

The molecular signaling pathways mediating human germ cell tumor (GCT) formation and progression are poorly understood despite a large number of studies detailing recurrent cytogenetic abnormalities. Germ cell tumors consist of multiple histologic subtypes and can also be divided into infantile/childhood or adolescent/adult tumors as well as gonadal or nongonadal sites of origin. All of these parameters are important in defining clinical outcome and in understanding the pathogenesis of these tumors. We utilized complementary DNA (cDNA) microarray analysis to identify differences in signal transduction pathways between 2 histologic subtypes of malignant ovarian GCTs (dysgerminomas versus ovarian endodermal sinus tumors). Hierarchical cluster analysis using only the genes involved in Wnt/beta-catenin signaling was able to distinguish these 2 tumor subtypes from each other. Wnt13 and beta-catenin showed significant differential expression patterns between the 2 tumor subtypes, and the results were confirmed by semiquantitative reverse transcriptase-polymerase chain reaction. Additional GCTs were studied for the expression of other members of Wnt/beta-catenin signaling, including Wnt13, frizzled, disheveled, low-density lipoprotein receptor-related protein 6, and beta-catenin. Differential expression levels were identified for several histologic subtypes of human GCTs. Finally, we prepared tissue microarrays containing GCTs from 83 different patients and demonstrated high levels of beta-catenin protein expression in 100% and nuclear accumulation in approximately 50% to 70% of all endodermal sinus tumors and immature teratomas (ITs). This pattern was independent of the patient's age. No nuclear accumulation of beta-catenin was observed in germinomas, embryonal carcinomas, or choriocarcinomas. These results indicate that activation of Wnt/beta-catenin signaling plays an important role in the pathogenesis of 2 histologic subtypes of human GCTs.

Since the discovery of testicular carcinoma in situ (CIS) -- the precursor cell for the vast majority of germ cell tumours -- it has been proposed that CIS cells could be derived from transformed primordial germ cells or gonocytes. Here, we review recent discoveries not only substantiating that initial hypothesis but also indicating that CIS cells have a striking phenotypic similarity to embryonic stem cells (ESC). Many cancers have been proposed to originate from tissue-specific stem cells [so-called 'cancer stem cells' (CSC)] and we argue that CIS may be a very good example of a CSC, but with exceptional features due to the retention of embryonic pluripotency. In addition, considering the fact that pre-invasive CIS cells are transformed from early fetal cells, possibly due to environmentally induced alterations of the niche, we discuss potential risks linked to the uncontrolled therapeutic use of ESC.

Objective: To study the immunohistochemical expression of OCT4, CD117 and CD30 in germ cell tumors and to assess their diagnostic value., Methods: Immunohistochemical study for OCT4 was performed on formalin-fixed, paraffin-embedded tissues of 63 cases of germ cell tumors, including seminoma (21), dysgerminoma (7), germinoma (8), embryonal carcinoma (8), yolk sac tumor (6), mature teratoma (10) and immature teratoma (3), as well as 25 cases of non-germ cell tumors, including granulosa cell tumor (8), clear cell adenocarcinoma (4), Leydig's cell tumor (5), diffuse large B-cell lymphoma (4) and malignant melanoma (4). Besides, the expression of CD117 and CD30 in all germ cell tumors was studied., Results: All cases of seminoma and germinoma, 6/7 cases of dysgerminoma and 7/8 cases of embryonal carcinoma were positive for OCT4, with strong nuclear staining. All other germ cell tumors and non-germ cell tumors were negative for OCT4, except for 1 case of yolk sac tumor and 1 case of clear cell adenocarcinoma which showed weak staining. Positive membranous expression of CD117 was demonstrated in 19/21(90.5%) seminoma, 5/7 dysgerminoma and 7/8 germinoma. Focal weak membranous staining was also noted in 1 case of yolk sac tumor. The melanocytes in teratoma were also positive for CD117. All cases of embryonal carcinoma were negative. On the other hand, positive membranous expression of CD30 were demonstrated in 6/8 embryonal carcinoma. One case of germinoma and 1 case of yolk sac tumor showed weak cytoplasmic positivity. All cases of seminoma and dysgerminoma, 7/8 germinoma and all cases of teratoma were negative for CD30., Conclusions: OCT4 is a sensitive and relatively specific marker for diagnosing seminoma, dysgerminoma, germinoma and embryonal carcinoma. CD117 and CD30 immunostains, when used in combination, represent valuable tools for distinguishing embryonal carcinoma and seminoma, dysgerminoma, germinoma.

In our immunohistochemical study of 25 human primary intracranial germinomas and germinomas with syncytiotrophoblastic giant cells (STGC), we stained the same sections for c-kit and placental alkaline phosphatase (PLAP). Immunohistochemical expression was graded using a semi-quantitative scoring system where 3+ =51-75%, and 4+ =76-100%. Of the 25 cases, 7 (28%) were graded 3+ and 18 (72%) 4+ for c-kit; 8 (32%) were 3+ or 4+ for PLAP. All 3 cases negative for PLAP-staining were strongly positive and all embryonal carcinomas, immature teratomas, and yolk sac tumors were negative for c-kit staining. The soluble isoform of c-kit (s-kit) is reportedly detectable in cerebral spinal fluid of patients with germinomas and germinomas with STGC. C-kit and s-kit may be powerful tumor markers for germinomas with or without STGC.

Context: Maturation delay of germ cells and their progression into carcinoma in situ (CIS) frequently occurs in intersex patients. A developmentally delayed germ cell resembles a CIS cell and displays prolonged expression of immunohistochemical markers used for the diagnosis of CIS. This questions their applicability in young children., Objective: The objective of the study was the elaboration of tools to distinguish germ cells with maturation delay and CIS., Design: The design was a qualitative and quantitative analysis of the expression of diagnostic markers for CIS in gonads of young patients with undervirilization syndromes., Setting: The study was conducted in the pathology department of a university center, specializing in germ cell tumor pathogenesis., Patients: Fifty-eight formalin-fixed, paraffin-embedded testicular tissue samples of 30 undervirilized patients (1 month to 23 yr of age) were analyzed., Interventions: INTERVENTIONS included hematoxylin-eosin staining, immunohistochemistry for octamer binding transcription factor (OCT)3/4, gene encoding the stem cell factor receptor that has tyrosine kinase activity c-KIT, placental/germ alkaline phosphatase (PLAP), testis-specific protein Y encoded (TSPY), and VASA, double staining for OCT3/4 and VASA, with ploidy determination by fluorescent in situ hybridization., Main Outcome Measure: Maturation delay and CIS are characterized by the staining patterns of the immunohistochemical markers., Results: CIS was diagnosed in three of 30 patients (10%) and four of 58 gonads (6.9%). Patient age, distribution of OCT3/4-positive cells throughout the gonad, and their position within the seminiferous tubule differ between maturation delay and CIS. Abnormal OCT3/4 and testis-specific protein Y encoded expression appear to be of pathogenetic relevance in the development of these lesions., Conclusion: The dimorphic expression of OCT3/4 allows distinction between maturation delay and CIS. Studies in larger patient series are essential before a biopsy to evaluate the neoplastic risk can eventually be proposed as an alternative for gonadectomy.

Some patients with testicular cancer exhibit low human chorionic gonadotropin (hCG) levels even after successful treatment. We report 2 patients with testicular cancer (2 non-seminomatous germ cell tumors [NSGCTs]) and low betahhCG levels with no recurrence after successful treatment. The first patient is a 45-year-old man with pulmonary and retroperitoneal lymph node metastases. During the first chemotherapy the hCG level decreased but remained at a low level. Salvage chemotherapy had no effect on the hCG level, but a low betahCG level persisted for 16 months. The second case is a 19-year-old man with testicular cancer with retroperitoneal lymph node metastases. The patient underwent an operation for residual masses after chemotherapy. Pathological findings showed necrotic tissue, and a low betahCG level persist for 160 months. A low hCG level does not necessarily meen residual malignancy. Patients with a low hCG level should be monaged carefully avoiding needless treatment.

Aims: NANOG is a key regulator of embryonic stem cell (ESC) self-renewal and pluripotency. Our recent genome-wide gene expression profiling study of the precursor of testicular germ cell tumours, carcinoma in situ testis (CIS), showed close similarity between ESC and CIS, including high NANOG expression. In the present study we analysed the protein expression of NANOG during normal development of human testis and in a large series of neoplastic/dysgenetic specimens., Methods and Results: We detected abundant expression of NANOG in CIS and in CIS-derived testicular tumours with marked differences; seminoma and embryonal carcinoma were strongly positive, differentiated somatic elements of teratoma were negative. We provide evidence for the fetal origin of testicular cancer as we detected strong expression of NANOG in fetal gonocytes up to gestational week 20, with subsequent down-regulation occurring earlier than for OCT-4. We detected no expression at the protein level in normal testis., Conclusions: NANOG is a new marker for testicular CIS and germ cell tumours and the high level of NANOG along with OCT-4 are determinants of the stem cell-like pluripotency of the preinvasive CIS cell. Timing of NANOG down-regulation in fetal gonocytes suggests that NANOG may act as a regulatory factor up-stream to OCT-4.