Intratubular Germ Cell Neoplasia Unclassified Research Articles

Analysis of POU5F1, c-Kit, PLAP, AP2γ and SALL4 in gonocytes of patients with cryptorchidism

Cryptorchidism is a risk factor for the development of testicular germ cell tumors (TGCTs). The most common type of TGCT in cryptorchidism is seminoma. The intratubular germ cell neoplasia unclassified (ITGCNU) is a histological pattern preceding the development of seminomas and non-seminomas. It was suggested that in patients with cryptorchidism, the gonocytes remained undifferentiated with pluripotent abilities expressing proteins like POU domain class 5 transcription factor 1 (POU5F1), tyrosine kinase receptor c-Kit, placental-like alkaline phosphatase (PLAP), the transcription factor AP2γ and sal-like protein 4 (SALL4) that confer to the gonocytes this ability and therefore make them susceptible to develop ITGCNU. The aim of the present study was to determine if the gonocytes of patients with cryptorchidism express POU5F1, c-Kit, PLAP, AP2γ and SALL4 proteins after their differentiation period. Based on this, we evaluated samples of testicular tissue from newborns to 16-year old subjects with or without cryptorchidism in search of POU5F1, c-Kit, PLAP, AP2γ and SALL4 using immunocytochemical method, the results of which were validated by RT-PCR. The results showed that control subjects witnessed a down-regulation in the expression of these five proteins in the first year of life, which eventually disappeared. On the other hand, it was determined that 21.6% (8/37) of the patients with cryptorchidism continued to express, at least, one of the proteins analyzed in this study after the second year of life. And only 5.4% (2/37) of the patients were positive to the five markers. These data sustain the proposed hypothesis that in cryptorchid patients, ITGCNU arises from gonocytes that fail in their differentiation process to spermatogonia with conservation of the proteins (POU5F1, c-Kit, PLAP, AP2γ and SALL4) that maintain pluripotency and undifferentiated characteristics and which are responsible for making the gonocytes susceptible to malignancy. However, we cannot guarantee that these patients present neoplastic transformation.

Frequent PD-L1 expression in testicular germ cell tumors.

Background:Many testicular germ cell cancers are curable despite metastatic disease, but about 10–15% of patients fail cisplatin-based first-line treatment. Immunotherapy is considered as additional treatment approach for these patients. Inhibition of the interaction between Programmed Death Receptor 1 (PD-1) and Programmed Death Receptor Ligand 1 (PD-L1) enhances T-cell responses in vitro and mediates clinical antitumour activity. We analysed the expression of PD-L1 in testicular germ cell tumours to evaluate its potential as target for immunotherapeutic strategies.Methods:Immunohistochemistry was performed in 479 formalin-fixed paraffin-embedded specimens using a rabbit monoclonal antibody (E1L3N). The tissue microarray consisted of 208 pure seminomas, 121 non-seminomas, 20 intratubular germ cell neoplasia unclassified (IGCNU) and 20 specimens of non-neoplastic testicular tissue.Results:Programmed Death Receptor Ligand-1 expression was found in 73% of all seminomas and in 64% of all non-seminomas. None of 20 IGCNU and none of 20 normal tissue specimens exhibited PD-L1 expression. PD-L1 positive stromal cells were only detected in seminomas, but not in non-seminomas. The anti PD-L1 antibody showed a pre-dominantly membranous staining pattern in testicular tumour cells, as well as expression in stromal cells.Conclusions:This frequent expression of PD-L1 in human testicular germ cell tumours suggests that patients with testicular germ cell tumours could profit from immunotherapeutic strategies using anti-PD1 and anti-PDL1 antibodies.

Intratubular germ cell neoplasia of the testis: a brief review.

Germ cell tumors of the testis may be divided into 3 broad categories according to age at presentation. The tumors in the pediatric age group include teratoma and yolk sac tumor. These tumors are generally not associated with convincing intratubular neoplasia. The second group consists of tumors presenting in third and fourth decade of life and include seminoma, embryonal carcinoma, yolk sac tumor, choriocarcinoma, and teratoma as well as mixed germ cell tumors. The precursor cell for these tumors is an abnormal gonocyte that fails to differentiate completely into spermatogonia. These abnormal cells stay dormant in the gonad during intrauterine life as well as infancy and childhood, but undergo proliferation during puberty and can be identified as intratubular germ cell neoplasia unclassified (IGCNU). These tumor cells continue to manifest protein expression pattern that resembles primitive germ cells (PLAP, c-KIT, OCT3/4). After a variable interval following puberty, IGCNU cells may acquire ability to penetrate the seminiferous tubules and present as an overt germ cell tumor. Acquisition of isochrome 12 and other genetic abnormalities are usually associated with this transition. The level of DNA methylation generally determines the phenotype of the germ cell tumor. The third type of germ cell tumors is spermatocytic seminoma, which is a rare tumor encountered later in life usually in fifth and sixth decade. The cell of origin of this tumor is probably postpubertal mature spermatogonia which acquire abnormal proliferative capability probably due to gain of chromosome 9 resulting in activation and amplification of genes such as DMRT1. The tumor cells manifest many of the proteins normally expressed by mature sperms such as VASA, SSX2, and occasionally OCT2. Although spermatocytic seminoma may also have an intratubular growth phase, it completely lacks features of IGCNU.

Frequent expression of PD-L1 in testicular germ cell tumors.

379 Background: Testicular germ-cell cancer is curable even in the presence of metastatic disease. Yet, about 10-15% of patients become cisplatin-refractory and will eventually die of their disease. Moreover, short- and long-term side effects of cisplatin make the search for less toxic treatment strategies worthwile. Programmed Death Receptor 1 (PD-1, CD279) is one member of the extended family of T cell regulators expressed on the surface of activated T cells, B cells, and macrophages. Its ligand, PD-L1 (B7-H1, CD274), is expressed on tumor cells, T cells and other tissues. The interaction of these two molecules negatively regulates immune responses. Of major interest is that inhibition of the interaction between PD-1 and PD-L1 can enhance T-cell responses in vitro and mediate clinical antitumor activity . The aim of this study was to analyze the expression of PD-L1 in testicular germ-cell tumors to evaluate its potential as predictive marker for further therapeutic strategies. Methods: Immunohistochemistry was performed in 486 Formalin Fixed Paraffin Embedded (FFPE) specimens using a monoclonal rabbit antibody (E1L3N, Cell Signaling Technology, Inc. (CST) of Danvers, MA, USA). Results: PD-L1 expression was found in 171 out of 248 (69%) seminomas, 19 out of 48 (40%) yolk sac tumors, 7 out of 46 (15%) teratomas, 2 out of 10 (20%) choriocarcinomas and 53 out of 87 (61%) embryonal carcinomas. In 20 out of 20 Intratubular germ-cell neoplasia unclassified (IGCNU) and also in 20 out of 20 normal tissue specimens no single case exhibited PD-L1 expression. Conclusions: Our study describes for the first time the frequent expression of PD-L1 in a large series of human testicular germ-cell tumors, but not on normal testis tissue or IGCNU. Based on our results, checkpoint inhibition with anti-PD1 and anti-PDL1 antibodies might represent an attractive approach in germ-cell cancer, where new active agents are urgently needed.

Gonadoblastoma: an immunohistochemical study and comparison to Sertoli cell nodule with intratubular germ cell neoplasia, with pathogenetic implications.

To investigate the immunohistochemical properties of the sex cord cells of gonadoblastoma and Sertoli cell nodule with intratubular germ cell neoplasia unclassified (IGCNU) as a means of objective distinction and to provide insight into the pathogenesis. Immunohistochemical stains for SOX9, FoxL2 and SF-1 were performed on 10 gonadoblastomas (all phenotypical females) and 14 Sertoli cell nodules with IGCNU in normal phenotypical males with coexisting germ cell tumours. The sex cord cells of gonadoblastomas showed strong, diffuse FoxL2 and SF-1 positivity and focal weak to moderate SOX9 reactivity, whereas those of Sertoli cell nodules with IGCNU were uniformly, strongly positive for SOX9 and SF-1, while negative for FoxL2. Coexpression of SOX9 and FoxL2 in the sex cord cells of gonadoblastomas provides evidence that these morphologically ambiguous sex cord cells are incompletely differentiated. The strong, diffuse SOX9 and SF-1 positivity and absence of FoxL2 reactivity in the Sertoli cell nodules with IGNCU support full Sertoli cell differentiation of the sex cord cells and distinguish them from gonadoblastomas. Deficient SOX9 expression in gonadoblastoma supports a current model of pathogenesis where immature germ cells, in the absence of well-formed Sertoli cells, retain a fetal phenotype and susceptibility to malignant transformation.

Idiopathic granulomatous orchitis: morphology and evaluation of its relationship to IgG4 related disease

Idiopathic granulomatous orchitis (IGO) is rare, thought to result from an autoimmune reaction to spermatogenic elements. Its relationship to IgG4-related disease (IgG4-RD) has not been evaluated. Sixteen orchiectomy specimens (1984-2012) with a prominent intratubular granulomatous reaction were reviewed: IGO (n = 6); intratubular germ cell neoplasia unclassified (IGCNU) with a granulomatous reaction and associated seminoma (GS, n = 6); and unclassified intratubular granulomatous orchitis not fitting into a specific entity (UGO, n = 4). Men with IGO were 32 to 86 years old, presenting with a mass suspicious for malignancy. Only one patient had a history of an inflammatory disease. Clinical follow-up was available for 2 patients with IGO, and both had no evidence of systemic IgG4-RD. All IGO cases had an epithelioid granulomatous reaction confined to seminiferous tubules, an extensive interstitial lymphoplasmacytic inflammation, 3 of 6 had prominent interstitial fibrosis, and 3 of 6 cases had plasma cells with an IgG4+/IgG+ ratio >40%. In GS, 10% to 100% of tubules with IGCNU had a granulomatous reaction, which in 3 cases replaced IGCNU cells. In contrast to IGO, GS had more intratubular multinucleated giant cells, more peritubular sclerosis, fewer interstitial plasma cells, and no interstitial fibrosis. Of the 4 UGO cases, most had predominantly interstitial with less intratubular granulomatous inflammation. Only 1 non-IGO case had elevated tissue IgG4 (GS case). It is critical and sometimes difficult to distinguish GS from IGO. IGO shares some features with IgG4-RD, yet current evidence does not support its classification as a localized manifestation of IgG4-RD occurring in the testis.

Just another brick in the wall: unravelling the epigenetic mechanism underlying tumour initiation of testicular germ cell cancer

Clarifying the genomic basis of human diseases and its many breakthroughs in cancer medicine has distracted our view from the importance and complexity of epigenomic information. A wall of dogmas awaiting their destruction was built: DNA methylation was proposed to occur passively but subsequently active DNA demethylation was discovered (Cedar, 1988). Histone methylation was thought to be irreversible until the first histone methylase, lysine-specific demethylase 1, was discovered and shown to provide essential cues for transcriptional regulation (Shi et al, 2004; Metzger et al, 2005). Subsequently, understanding the epigenome and its key writer enzymes, methylases and demethylases, provided important insights into pathomechanisms of aging, cancer and inflammation. The current study by Kristensen et al (2014) now closes another gap by providing evidence for an epigenetic mechanism driving intratubular germ cell neoplasia unclassified (ITGCNU), the precursor lesion for invasive testicular germ cell cancer (TGCC).

RETRACTED ARTICLE: Critical role of CCDC6 in the neoplastic growth of testicular germ cell tumors

BackgroundDNA damage response has been clearly described as an anti-cancer barrier in early human tumorigenesis. Moreover, interestingly, testicular germ cell tumors (TGCTs) have been reported to lack the DNA Damage Response (DDR) pathway activation.CCDC6 is a pro-apoptotic phosphoprotein substrate of the kinase ataxia telangectasia mutated (ATM) able to sustain DNA damage checkpoint in response to genotoxic stress and is commonly rearranged in malignancies upon fusion with different partners.In our study we sought to determine whether CCDC6 could have a role in the patho-genesis of testicular germ cell tumors.MethodsTo achieve this aim, analysis for CCDC6 expression has been evaluated on serial sections of the mouse testis by immunohistochemistry and on separate populations of murine testicular cells by western blot. Next, the resistance to DNA damage-induced apoptosis and the production of reactive oxygen species has been investigated in GC1 cells, derived from immortalized type B murine germ cells, following CCDC6 silencing. Finally, the CCDC6 expression in normal human testicular cells, in Intratubular Germ Cell Neoplasia Unclassified (IGCNU), in a large series of male germ cell tumours and in the unique human seminoma TCam2 cell line has been evaluated by immunohistochemistry and by Western Blot analyses.ResultsThe analysis of the CCDC6 expression revealed its presence in Sertoli cells and in spermatogonial cells. CCDC6 loss was the most consistent feature among the primary tumours and TCam2 cells. Interestingly, following treatment with low doses of H2O2, the silencing of CCDC6 in GC1 cells caused a decrease in the oxidized form of cytochrome c and low detection of Bad, PARP-1 and Caspase 3 proteins. Moreover, in the silenced cells, upon oxidative damage, the cell viability was protected, the γH2AX activation was impaired and the Reactive Oxygen Species (ROS) release was decreased.ConclusionsTherefore, our results suggest that the loss of CCDC6 could aid the spermatogonial cells to be part of a pro-survival pathway that helps to evade the toxic effects of endogenous oxidants and contributes to testicular neoplastic growth.

Allelotyping analysis suggesting a consecutive progression from intratubular germ cell neoplasia to seminoma and then to embryonal carcinoma of the adult testis

Among adult testicular germ cell tumors, the pathogenesis of embryonal carcinoma remains a matter of debate. Some studies suggest a single consecutive progression from intratubular germ cell neoplasia, unclassified (IGCNU), to seminoma and then to embryonal carcinoma; others suggest that seminoma and embryonal carcinoma derive independently from IGCNU. This allelotyping study aimed to clarify the genetic relationship between embryonal carcinoma components and coexisting seminoma and/or IGCNU components. From a cohort of 18 patients with embryonal carcinoma, 11 coexisting seminoma components and 14 coexisting IGCNUs were identified. DNA isolated from each laser-microdissected tissue was subjected to polymerase chain reaction and loss of heterozygosity (LOH) analysis, using 20 polymorphic markers located on 12 chromosome arms (3q, 5q, 6p, 9p, 10q, 11p, 12p, 12q, 13q, 17p, 17q, and 18q). The concordance rate for allelic patterns was 82% between IGCNU and the coexisting seminoma components, 71% between IGCNU and the coexisting embryonal carcinoma components, and 80% between seminoma components and the coexisting embryonal carcinoma components. Estimation of probability indicated that these events were very unlikely to have occurred by chance. The total frequency of LOH increased progressively from IGCNU to seminoma and then to embryonal carcinoma, with statistically significant differences. In 7 cases with 3 histologic components, 28 chromosomal loci that showed LOH in the seminoma and embryonal carcinoma components were identified, and 15 (54%) retained heterozygosity in the coexisting IGCNUs. These findings suggest that a consecutive progression from IGCNU to seminoma, and ultimately, to embryonal carcinoma mainly occurred in the testicular germ cell tumor cases.

Premalignancy of the testis and paratestis

There has been great progress in the understanding of the molecular events involved in normal germ cell maturation and testicular tumour pathobiology. Identification of the molecules involved in these processes has provided a number of new immunohistochemical markers for intratubular germ cell neoplasia, unclassified (IGCNU) and germ cell tumours. OCT4, the most important of the newer markers, is highly sensitive and specific for IGCNU and is superior to older immunohistochemical markers for this purpose. However, germ cell maturation delay is a potential pitfall in the application of this marker. In contrast to the type II testicular germ cell tumours, relatively little is known about premalignant lesions of testicular sex cord-stromal tumours, malignancies of the rete testis and epididymis, and paratesticular malignancies.

PARP expression in germ cell tumours

BackgroundPoly(ADP-ribose)polymerase (PARP) inhibitors represent a new class of promising drugs in anticancer therapy.AimsTo evaluate PARP expression in testicular germ cell tumours (GCTs) and to correlate expression patterns with clinicopathological variables.MethodsIn...

Probability of Metachronous Testicular Cancer in Patients With Biopsy-Proven Intratubular Germ Cell Neoplasia Depends on First-Time Treatment of Germ Cell Cancer

To evaluate the probability of subsequent testicular cancer (STC) in patients with intratubular germ cell neoplasia unclassified (IGCNU) treated for first-time invasive germ cell cancer. Sixty-one patients with germ cell testicular cancer or extragonadal germ cell cancer received follow-up from diagnosis of IGCNU to development of STC, initiation of IGCNU-definitive treatment (orchiectomy/radiotherapy), emigration, death, or end of follow-up. The probability of STC was assessed in subgroups according to chemotherapy burden. The probability of STC in the nonexposed patients was significantly increased compared with those exposed to chemotherapy (P = .05; 5-year probability of 54% [95% CI, 33% to 78%] and 23% [95% CI, 11% to 45%], respectively). In the group of patients treated with one to three cycles or no chemotherapy, the probability of STC was significantly increased compared with those exposed to four or more cycles (P = .03; 5-year probability of 42% [95% CI, 27% to 62%] and 22% [95% CI, 8% to 54%], respectively). Twenty-two of 22 patients were tumor-free and alive at a median of 56 months (range, 2 to 184 months) after diagnosis of STC. Platinum-based chemotherapy may reduce the probability of STC in patients with IGCNU, particularly in those treated with four or more cycles of chemotherapy. A watch-and-wait strategy for patients with IGCNU may be justified in selected patients with future plans for paternity.

Protein overexpression and gene amplification of epidermal growth factor receptor in adult testicular germ cell tumors: Potential role in tumor progression

Little is known about the pathologic significance of epidermal growth factor receptor (EGFR) expression in malignant testicular germ cell tumors (TGCTs) in adults. From the primary tumor sites of a cohort of 110 TGCT cases, we obtained 209 histologically distinct components: 53 intratubular germ cell neoplasia unclassified (IGCNU) lesions, 83 seminomas (66 pure-form seminomas and 17 seminoma components in the mixed-form with nonseminomatous TGCTs), 27 embryonal carcinomas, eight choriocarcinomas, 18 yolk sac tumors, and 20 immature teratomas. Samples were analyzed for expression of EGFR protein and EGFR gene amplification by immunohistochemistry and fluorescence in situ hybridization (FISH), respectively. Overexpression of the EGFR protein was detected in 28% of seminomas (27% in the pure-form and 29% in the mixed-form), 11% of embryonal carcinomas, 88% of choriocarcinomas, 44% of yolk sac tumors, and none of the IGCNU lesions or immature teratomas. A higher copy number (≥4 copies per cell) and amplification of the EGFR gene were detected in 20% and 10% of seminomas, 13% and 0% of embryonal carcinomas, 71% and 60% of choriocarcinomas, 15% and 8% of yolk sac tumors, and none of the IGCNU lesions or immature teratomas, respectively. Both higher copy number and amplification of the EGFR gene were positively correlated with immunohistochemical overexpression of EGFR protein (each P < 0.0001). These results suggest that overexpression of EGFR protein and increased copy number or amplification of the EGFR gene occur relatively frequently in primary TGCTs, and may play roles in the formation of invasive cancer and in the progression, especially morphological evolution, of tumors.

The pluripotency transcription factor Kruppel-like factor 4 is strongly expressed in intratubular germ cell neoplasia unclassified and seminoma

Germ cell tumors of the testis are the most frequent tumors in men between 20 and 40 years. Their most common subtype is the seminoma, which arises like the embryonal carcinoma from an intratubular germ cell neoplasia unclassified (IGCNU), i.e. fetal germ cells that escaped from the control of the developing testicular stem cell niche, eventually leading to a fully developed seminoma (or embryonal carcinoma). The molecular causes for the development of an IGCNU are still unknown. However, IGCNU cells share the expression of several factors with primordial germ cells and gonocytes and, interestingly, also with pluripotent embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. One factor playing important roles in both iPS and ES cells is the transcription factor Krüppel-like factor 4 (KLF4). This study examined KLF4 expression data from 179 human testicular samples including normal controls and seminoma, deposited in Gene Expression Omnibus repository for microarray data at the National Centre for Biotechnology Information. Immunohistochemistry was used to detect KLF4 protein expression in IGCNU (n = 6), seminoma (n = 14) and fetal human testes (n = 14). Microarray data from three independent sources suggest higher mRNA expression in seminoma than in normal testis. Normal spermatogonia, which are the stem cells of spermatogenesis, controlled by their stem cell niche, do not express KLF4. In contrast, IGCNU and seminoma cells strongly express KLF4. In conclusion, this finding suggests that KLF4 may be an important factor for the maintenance of the developmental and the tumorigenic potential of IGCNU as well as for the malignancy of seminoma.

Global DNA hypomethylation in intratubular germ cell neoplasia and seminoma, but not in nonseminomatous male germ cell tumors

Alterations in methylation of CpG dinucleotides at the 5 position of deoxycytidine residues (5mC) are a hallmark of cancer cells, including testicular germ cell tumors. Virtually all testicular germ cell tumors are believed to be derived from intratubular germ cell neoplasia unclassified (IGCNU), which is thought to arise from primordial germ cells. Prior studies revealed that seminomas contain reduced levels of global DNA methylation as compared with nonseminomatous germ cell tumors. Smiraglia et al have proposed a model whereby seminomas arise from IGCNU cells derived from primordial germ cells that have undergone 5mC erasure, and nonseminomas arise from IGCNU cells derived from primordial germ cells that have already undergone de novo methylation after the original erasure of methylation and contain normal 5mC levels. Yet the methylation status of IGCNU has not been determined previously. We used immunohistochemical staining against 5mC to evaluate global methylation in IGCNU and associated invasive testicular germ cell tumors. Strikingly, staining for 5mC was undetectable (or markedly reduced) in the majority of IGCNU and seminomas, yet there was robust staining in nonseminomatous germ cell tumors. The lack of staining for 5mC in IGCNU and seminomas was also found in mixed germ cell tumors containing both seminomatous and nonseminomatous components. Lack of 5mC staining was not related to a lack of the maintenance methyltransferase (DNA methyltransferase 1) protein. We conclude that testicular germ cell tumors are derived in most cases from IGCNU cells that have undergone developmentally programmed 5mC erasure and that the degree of subsequent de novo methylation is most closely related to the differentiation state of the neoplastic cells. That is, IGCNU cells and seminoma cells remain unmethylated, whereas all other histological types appear to arise after de novo methylation.

Heterogeneous Distribution of ITGCNU in an Adult Testis: Consequences for Biopsy-Based Diagnosis

Carcinoma in situ (CIS) of the testis, also referred to as intratubular germ cell neoplasia unclassified (ITGCNU), is currently accepted as the common precursor for all malignant germ cell tumors of adolescents and adults- that is, the seminomatous and nonseminoma cancers. These preinvasive cells have specific cellular characteristics, which can be used for the early diagnosis-routinely done by morphological analysis, sometimes supported by immunohistochemistry-of tissue obtained by an open surgical biopsy. False-negative biopsy results can occur mostly because of the nonrandom distribution of ITGCNU within the testis, misdiagnosis, or suboptimal tissue treatment and analysis. In this article, we demonstrate the potential pitfalls in the diagnosis of ITGCNU. The results support the use of the highly specific and sensitive immunohistochemical marker OCT3/4 for the diagnosis of ITGCNU and provide evidence for the nonrandom distribution of ITGCNU, which is a significant limitation in the diagnosis of this preinvasive lesion.

Chromosomes and Expression in Human Testicular Germ‐Cell Tumors

Human germ-cell tumors (GCTs) are a heterogeneous group of neoplasms. Based on epidemiology, anatomical site of presentation, histology, chromosomal constitution, and pattern of genomic imprinting, GCTs are classified into five entities. Within the testis, three types of GCTs can be diagnosed: type I (teratomas and yolk-sac tumors of neonates and infants); type II (seminomas and nonseminomas); type III (spermatocytic seminomas). Here the focus is on the type II GCTs, the most frequent type in the adult testis (so-called TGCTs). They can also be diagnosed in dysgenetic gonads (an incomplete or defective formation of the gonad, caused by a disturbed process of migration of the germ cells and/or their correct organization in their fetal gonadal ridge), the anterior mediastinum, and pineal/suprasellar region. In the testis, they originate from the malignant counterpart of primordial germ cells/gonocytes, referred to as carcinoma in situ (CIS)/intratubular germ-cell neoplasia unclassified (ITGCNU). CIS/ITGCNU and seminomatous cells are characterized by expression of OCT3/4 and NANOG, while in addition embryonal carcinoma expresses SOX2, all identified as transcription factors related to pluripotency in embryonic stem (ES) cells. With the exception of teratomas, most histological elements of TGCTs are sensitive for (cisplatin-based) chemotherapy; CIS/ITGCNU and seminoma cells are also sensitive to DNA damage induced by irradiation. Similar observations have been made for ES cells and their derivates. Moreover, the genetic constitution of TGCTs (low incidence of mutations and frequent uniparental disomies) can also be linked to characteristics of ES cells, likely related to their specific inability to repair DNA damage and their high sensitivity to apoptotic cell death. The unusual presence of wild-type P53 in TGCTs is explained by specific expression of a cluster of micro-RNAs (miRNAs), that is, hsa-miR 371-373, also expressed in ES cells, which prevents P53-driven cellular senescence upon oncogenic stress. Many characteristics of human TGCTs reflect the nonmalignant counterparts from which they originate. Demonstration of these characteristics, in combination with the knowledge of the abnormal niche of these cells, normally occupied by spermatogonia, allows an informative method for (early) diagnosis. The conclusion is that TGCTs are embryonic cancers found in adults.

Tumor risk in disorders of sex development (DSD)

Disorders of sex development (DSD), previously referred to as intersex disorders, comprise a variety of anomalies defined by congenital conditions in which chromosomal, gonadal, or anatomical sex is atypical. Besides issues such as gender assignment, clinical and diagnostic evaluation, surgical and psychosocial management, and sex steroid replacement, the significantly increased risk for developing specific types of malignancies is both clinically and biologically relevant. This relates to germ-cell tumors specifically in DSD patients with hypovirilization or gonadal dysgenesis. The presence of a well-defined part of the Y chromosome (known as the GBY region) is a prerequisite for malignant transformation, for which the testis-specific protein on the Y chromosome (TSPY) is a likely candidate gene. The precursor lesions of these cancers are carcinoma in situ (CIS)/intratubular germ-cell neoplasia unclassified (ITGCNU) in testicular tissue and gonadoblastoma in those without obvious testicular differentiation. Most recently, undifferentiated gonadal tissue (UGT) has been identified as the likely precursor for gonadoblastoma. The availability of markers for the different developmental stages of germ cells allows detailed investigation of the characteristics of normal and (pre)malignant germ cells. Although informative in a diagnostic setting for adult male patients, these markers - such as OCT3/4 - cannot easily distinguish (pre)malignant germ cells from germ cells showing delayed maturation. This latter phenomenon is frequently found in gonads of DSD patients, and may be related to the risk of malignant transformation. Thus, the mere application of these markers might result in over-diagnosis and unnecessary gonadectomy. It is proposed that morphological and histological evaluation of gonadal tissue, in combination with OCT3/4 and TSPY double immunohistochemistry and clinical parameters, is most informative in estimating the risk for germ-cell tumor development in the individual patient, and might in future be used to develop a decision tree for optimal management of patients with DSD.

OCT4: biological functions and clinical applications as a marker of germ cell neoplasia

Germ cell tumours (GCTs) are a heterogeneous group of neoplasms, which develop in the gonads as well as in extragonadal sites, that share morphological patterns and an overall good prognosis, owing to their responsiveness to current surgical, chemotherapeutic, and radiotherapeutic measures. GCTs demonstrate extremely interesting biological features because of their close relationships with normal embryonal development as demonstrated by the pluripotentiality of some undifferentiated GCT variants. The similarities between GCTs and normal germ cell development have made it possible to identify possible pathogenetic pathways in neoplastic transformation and progression of GCTs. Genotypic and immunophenotypic profiles of these tumours are also useful in establishing and narrowing the differential diagnosis in cases of suspected GCTs. Recently, OCT4 (also known as OCT3 or POU5F1), a transcription factor that has been recognized as fundamental in the maintenance of pluripotency in embryonic stem cells and primordial germ cells, has been proposed as a useful marker for GCTs that exhibit features of pluripotentiality, specifically seminoma/dysgerminoma/germinoma and embryonal carcinoma. The development of commercially available OCT4-specific antibodies suitable for immunohistochemistry on paraffin-embedded specimens has generated increasing numbers of reports of OCT4 expression in a wide variety of gonadal and extragonadal GCTs. OCT4 immunostaining has been shown to be a sensitive and specific marker for seminomatous/(dys)germinomatous tumours and in embryonal carcinoma variants of non-seminomatous GCTs, whether in primary gonadal or extragonadal sites or in metastatic lesions. Therefore, OCT4 immunohistochemistry is an additional helpful marker both in the differential diagnosis of specific histological subtypes of GCTs and in establishing a germ cell origin for some metastatic tumours of uncertain primary. OCT4 expression has also been reported in pre-invasive conditions such as intratubular germ cell neoplasia, unclassified (IGCNU) and the germ cell component of gonadoblastoma. Additionally, OCT4 immunostaining shows promise as a useful tool in managing patients known to be at high risk for the development of invasive GCTs.

Activation‐induced cytidine deaminase (AID) is expressed in normal spermatogenesis but only infrequently in testicular germ cell tumours

Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes in antigen-dependent B-cell maturation. SHM is not restricted to immunoglobulin gene loci, raising the possibility of a function for AID in other cell types. In this study, it is shown that AID is expressed in spermatocytes in the human testis. AID was mostly cytoplasmic but nuclear AID was also observed in a proportion of cells, in keeping with the DNA deamination model of AID function. Intratubular germ cell neoplasia unclassified (IGCNU), the precursor lesion of testicular cancers, was AID-negative. Seminomas also lacked AID expression. Nuclear and cytoplasmic AID expression was observed in three of 32 mixed non-seminomatous germ cell tumours. The results provide evidence for a physiological role for AID outside the immune system. AID expression in spermatocytes points to a role in meiosis. It remains uncertain whether AID may also contribute to the genetic aberrations characteristically found in testicular germ cell tumours. The consistent absence of detectable AID expression in atypical spermatogonia of IGCNU and its rare expression in germ cell tumours suggest that continued expression of AID is not involved in the pathogenesis of germ cell tumours.