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1.  Delayed Recognition of Disorders of Sex Development (DSD): A Missed Opportunity for Early Diagnosis of Malignant Germ Cell Tumors 
Disorders of sex development (DSD) are defined as a congenital condition in which development of chromosomal, gonadal or anatomical sex is atypical. DSD patients with gonadal dysgenesis or hypovirilization, containing part of the Y chromosome (GBY), have an increased risk for malignant type II germ cell tumors (GCTs: seminomas and nonseminomas). DSD may be diagnosed in newborns (e.g., ambiguous genitalia), or later in life, even at or after puberty. Here we describe three independent male patients with a GCT; two were retrospectively recognized as DSD, based on the histological identification of both carcinoma in situ and gonadoblastoma in a single gonad as the cancer precursor. Hypospadias and cryptorchidism in their history are consistent with this conclusion. The power of recognition of these parameters is demonstrated by the third patient, in which the precursor lesion was diagnosed before progression to invasiveness. Early recognition based on these clinical parameters could have prevented development of (metastatic) cancer, to be treated by systemic therapy. All three patients showed a normal male 46,XY karyotype, without obvious genetic rearrangements by high-resolution whole-genome copy number analysis. These cases demonstrate overlap between DSD and the so-called testicular dysgenesis syndrome (TDS), of significant relevance for identification of individuals at increased risk for development of a malignant GCT.
PMCID: PMC3272341  PMID: 22315593
2.  Establishment and Characterization of a new Human Extragonadal Germ Cell Line, SEM-1, and its Comparison With TCam-2 and JKT-1 
Urology  2013;81(2):464.e1-464.e9.
To describe the establishment and characterization of a human cell line, SEM-1, from a patient diagnosed with a mediastinal seminoma.
A small percentage of germ cell tumors develop as primary lesions in extragonadal sites, and the etiology of these tumors is poorly understood. Currently, only 2 cell lines from seminoma patients have been reported, JKT-1 and TCam-2, both derived from the testis. The cell line was characterized by heterotransplantation in Nude mice, cytogenetic studies, immunohistochemical and flow cytometry staining for germ cell tumor biomarkers, quantitative reverse-transcription polymerase chain reaction for cancer testis antigen expression, and BRAF mutation screening with quantitative polymerase chain reaction.
Characterization studies confirmed the human extragonadal seminoma origin of SEM-1 and demonstrated that it had more features in common with TCam-2 than JKT-1. Specifically, SEM-1 was positive for Sal-like protein 4 (SALL-4), activator protein-2γ (AP-2γ), and cytokeratin CAM5.2, and demonstrated heterogeneous expression of stem cell markers octamer-binding transcription factor 3/4, NANOG, c-KIT, SOX17, and SOX2. Cytogenetic analysis revealed a hypotriploid chromosome number, with multiple copies of 12p, but isochromosome 12p and the BRAF mutation V600E were not identified. The cell lines also did not contain the BRD4/NUT gene rearrangement [t(15,19)] seen in midline carcinomas nor did they contain overexpressed nuclear protein in testis (NUT) genes.
SEM-1 is the first cell line derived from an extragonadal germ cell tumor showing intermediate characteristics between seminoma and nonseminoma, and as such, is an important model to study the molecular pathogenesis of this malignancy.
PMCID: PMC4179199  PMID: 23374840
3.  Seminoma and Embryonal Carcinoma Footprints Identified by Analysis of Integrated Genome-Wide Epigenetic and Expression Profiles of Germ Cell Cancer Cell Lines 
PLoS ONE  2014;9(6):e98330.
Originating from Primordial Germ Cells/gonocytes and developing via a precursor lesion called Carcinoma In Situ (CIS), Germ Cell Cancers (GCC) are the most common cancer in young men, subdivided in seminoma (SE) and non-seminoma (NS). During physiological germ cell formation/maturation, epigenetic processes guard homeostasis by regulating the accessibility of the DNA to facilitate transcription. Epigenetic deregulation through genetic and environmental parameters (i.e. genvironment) could disrupt embryonic germ cell development, resulting in delayed or blocked maturation. This potentially facilitates the formation of CIS and progression to invasive GCC. Therefore, determining the epigenetic and functional genomic landscape in GCC cell lines could provide insight into the pathophysiology and etiology of GCC and provide guidance for targeted functional experiments.
This study aims at identifying epigenetic footprints in SE and EC cell lines in genome-wide profiles by studying the interaction between gene expression, DNA CpG methylation and histone modifications, and their function in the pathophysiology and etiology of GCC. Two well characterized GCC-derived cell lines were compared, one representative for SE (TCam-2) and the other for EC (NCCIT). Data were acquired using the Illumina HumanHT-12-v4 (gene expression) and HumanMethylation450 BeadChip (methylation) microarrays as well as ChIP-sequencing (activating histone modifications (H3K4me3, H3K27ac)). Results indicate known germ cell markers not only to be differentiating between SE and NS at the expression level, but also in the epigenetic landscape.
The overall similarity between TCam-2/NCCIT support an erased embryonic germ cell arrested in early gonadal development as common cell of origin although the exact developmental stage from which the tumor cells are derived might differ. Indeed, subtle difference in the (integrated) epigenetic and expression profiles indicate TCam-2 to exhibit a more germ cell-like profile, whereas NCCIT shows a more pluripotent phenotype. The results provide insight into the functional genome in GCC cell lines.
PMCID: PMC4041891  PMID: 24887064
4.  Role of SOX2 in the Etiology of Embryonal Carcinoma, Based on Analysis of the NCCIT and NT2 Cell Lines 
PLoS ONE  2014;9(1):e83585.
The transcription factor SOX2, associated with amongst others OCT3/4, is essential for maintenance of pluripotency and self-renewal of embryonic stem cells. SOX2 is highly expressed in embryonal carcinoma (EC), the stem cell component of malignant nonseminomatous germ cell tumors, referred to as germ cell cancer (GCC). In fact, OCT3/4 together with SOX2 is an informative diagnostic tool for EC in a clinical setting. Several studies support the hypothesis that SOX2 is a relevant oncogenic factor in various cancers and recently, SOX2 has been suggested as a putative therapeutic target for early stage EC. We demonstrate the presence of genomic amplification of SOX2 in an EC cell line, NCCIT, using array comparative genome hybridization and fluorescence in situ hybridization. Down-regulation of SOX2 by targeted siRNA provokes NCCIT cells towards apoptosis, while inhibition of OCT3/4 expression induced differentiation, with retained SOX2 levels. Mice pluripotent xenografts from NCCIT (N-NCCIT and N2-NCCIT) show a consistent SOX2 expression, in spite of loss of the expression of OCT3/4, and differentiation, with retained presence of genomic amplification. No SOX2 amplification has been identified in primary pure and mixed EC in vivo patient samples so far. The data presented in this study are based on a single EC cell line with a SOX2 amplification, with NT2 as control EC cell line, showing no profound induction of apoptosis upon SOX2 downregulation. The findings are of relevance to identify mechanisms involved in the pathogenesis of EC tumors, and support the model of SOX2-oncogene dependency of EC, which however, does not exclude induction of differentiation. This finding is likely related to the presence of wild type p53 in GCC, resulting in expression of downstream target genes, amongst others miR-34a, miR-145 and SOX2, associated to the unique sensitivity of GCC to DNA damaging agents.
PMCID: PMC3880257  PMID: 24404135
5.  Mutations in LRRC50 Predispose Zebrafish and Humans to Seminomas 
PLoS Genetics  2013;9(4):e1003384.
Seminoma is a subclass of human testicular germ cell tumors (TGCT), the most frequently observed cancer in young men with a rising incidence. Here we describe the identification of a novel gene predisposing specifically to seminoma formation in a vertebrate model organism. Zebrafish carrying a heterozygous nonsense mutation in Leucine-Rich Repeat Containing protein 50 (lrrc50 also called dnaaf1), associated previously with ciliary function, are found to be highly susceptible to the formation of seminomas. Genotyping of these zebrafish tumors shows loss of heterozygosity (LOH) of the wild-type lrrc50 allele in 44.4% of tumor samples, correlating with tumor progression. In humans we identified heterozygous germline LRRC50 mutations in two different pedigrees with a family history of seminomas, resulting in a nonsense Arg488* change and a missense Thr590Met change, which show reduced expression of the wild-type allele in seminomas. Zebrafish in vivo complementation studies indicate the Thr590Met to be a loss-of-function mutation. Moreover, we show that a pathogenic Gln307Glu change is significantly enriched in individuals with seminoma tumors (13% of our cohort). Together, our study introduces an animal model for seminoma and suggests LRRC50 to be a novel tumor suppressor implicated in human seminoma pathogenesis.
Author Summary
Testicular Germ Cell Tumors are frequently occurring tumors, affecting 1 in 500 individuals. Of this diverse group, the subtype seminoma is most prevalent and is the most common tumor type found in men aged 20–40 years of age. In contrast to other frequently occurring tumor types, there is very little information on the genetic components that form risk factors for seminoma. In this study we describe the unexpected finding that zebrafish carrying a heterozygous mutation in the lrrc50/dnaaf1 gene have a high incidence for testicular germ cell tumor formation. Detailed analysis suggests that these tumors resemble human seminoma. We therefore analyzed this gene in a subset of human seminoma samples and recovered mutations that were subsequently demonstrated to prohibit protein function. Seminomas were also previously found in family members of these patients, suggesting that a genetic component is the underlying cause. We thus identified a novel gene that can be considered a risk factor for human seminoma, and we describe an animal model system that is valuable for further seminoma research.
PMCID: PMC3627517  PMID: 23599692
6.  Prevalence of c-KIT Mutations in Gonadoblastoma and Dysgerminomas of Patients with Disorders of Sex Development (DSD) and Ovarian Dysgerminomas 
PLoS ONE  2012;7(8):e43952.
Activating c-KIT mutations (exons 11 and 17) are found in 10–40% of testicular seminomas, the majority being missense point mutations (codon 816). Malignant ovarian dysgerminomas represent ∼3% of all ovarian cancers in Western countries, resembling testicular seminomas, regarding chromosomal aberrations and c-KIT mutations. DSD patients with specific Y-sequences have an increased risk for Type II Germ Cell Tumor/Cancer, with gonadoblastoma as precursor progressing to dysgerminoma. Here we present analysis of c-KIT exon 8, 9, 11, 13 and 17, and PDGFRA exon 12, 14 and 18 by conventional sequencing together with mutational analysis of c-KIT codon 816 by a sensitive and specific LightCycler melting curve analysis, confirmed by sequencing. The results are combined with data on TSPY and OCT3/4 expression in a series of 16 DSD patients presenting with gonadoblastoma and dysgerminoma and 15 patients presenting pure ovarian dysgerminomas without DSD. c-KIT codon 816 mutations were detected in five out of the total of 31 cases (all found in pure ovarian dysgerminomas). A synonymous SNP (rs 5578615) was detected in two patients, one DSD patient (with bilateral disease) and one patient with dysgerminoma. Next to these, three codon N822K mutations were detected in the group of 15 pure ovarian dysgerminomas. In total activating c-KIT mutations were found in 53% of ovarian dysgerminomas without DSD. In the group of 16 DSD cases a N505I and D820E mutation was found in a single tumor of a patient with gonadoblastoma and dysgerminoma. No PDGFRA mutations were found. Positive OCT3/4 staining was present in all gonadoblastomas and dysgerminomas investigated, TSPY expression was only seen in the gonadoblastoma/dysgerminoma lesions of the 16 DSD patients. This data supports the existence of two distinct but parallel pathways in the development of dysgerminoma, in which mutational status of c-KIT might parallel the presence of TSPY.
PMCID: PMC3429439  PMID: 22937135
7.  A 46,XY Female DSD Patient with Bilateral Gonadoblastoma, a Novel SRY Missense Mutation Combined with a WT1 KTS Splice-Site Mutation 
PLoS ONE  2012;7(7):e40858.
Patients with Disorders of Sex Development (DSD), especially those with gonadal dysgenesis and hypovirilization are at risk of developing malignant type II germ cell tumors/cancer (GCC) (seminoma/dysgerminoma and nonseminoma), with either carcinoma in situ (CIS) or gonadoblastoma (GB) as precursor lesion. In 10–15% of 46,XY gonadal dysgenesis cases (i.e., Swyer syndrome), SRY mutations, residing in the HMG (High Mobility Group) domain, are found to affect nuclear transport or binding to and bending of DNA. Frasier syndrome (FS) is characterized by gonadal dysgenesis with a high risk for development of GB as well as chronic renal failure in early adulthood, and is known to arise from a splice site mutation in intron 9 of the Wilms’ tumor 1 gene (WT1). Mutations in SRY as well as WT1 can lead to diminished expression and function of SRY, resulting in sub-optimal SOX9 expression, Sertoli cell formation and subsequent lack of proper testicular development. Embryonic germ cells residing in this unfavourable micro-environment have an increased risk for malignant transformation. Here a unique case of a phenotypically normal female (age 22 years) is reported, presenting with primary amenorrhoea, later diagnosed as hypergonadotropic hypogonadism on the basis of 46,XY gonadal dygenesis with a novel missense mutation in SRY. Functional in vitro studies showed no convincing protein malfunctioning. Laparoscopic examination revealed streak ovaries and a normal, but small, uterus. Pathological examination demonstrated bilateral GB and dysgerminoma, confirmed by immunohistochemistry. Occurrence of a delayed progressive kidney failure (focal segmental glomerular sclerosis) triggered analysis of WT1, revealing a pathogenic splice–site mutation in intron 9. Analysis of the SRY gene in an additional five FS cases did not reveal any mutations. The case presented shows the importance of multi-gene based diagnosis of DSD patients, allowing early diagnosis and treatment, thus preventing putative development of an invasive cancer.
PMCID: PMC3399878  PMID: 22815844
8.  A genome-wide RNAi screen in mouse embryonic stem cells identifies Mp1 as a key mediator of differentiation 
The Journal of Experimental Medicine  2011;208(13):2675-2689.
Knockdown of the scaffolding protein Mek binding protein 1 (Mp1) in mouse embryonic stem cells suppresses differentiation but not proliferation, and more invasive human germ cell tumors express lower amounts of Mp1.
Despite intense investigation of intrinsic and extrinsic factors that regulate pluripotency, the process of initial fate commitment of embryonic stem (ES) cells is still poorly understood. We used a genome-wide short hairpin RNA screen in mouse ES cells to identify genes that are essential for initiation of differentiation. Knockdown of the scaffolding protein Mek binding protein 1 (Mp1, also known as Lamtor3 or Map2k1ip1) stimulated self-renewal of ES cells, blocked differentiation, and promoted proliferation. Fibroblast growth factor 4 (FGF4) signaling is required for initial fate commitment of ES cells. Knockdown of Mp1 inhibited FGF4-induced differentiation but did not alter FGF4-driven proliferation. This uncoupling of differentiation and proliferation was also observed when oncogenic Ras isoforms were overexpressed in ES cells. Knockdown of Mp1 redirected FGF4 signaling from differentiation toward pluripotency and up-regulated the pluripotency-related genes Esrrb, Rex1, Tcl1, and Sox2. We also found that human germ cell tumors (GCTs) express low amounts of Mp1 in the invasive embryonic carcinoma and seminoma histologies and higher amounts of Mp1 in the noninvasive carcinoma in situ precursor and differentiated components. Knockdown of Mp1 in invasive GCT cells resulted in resistance to differentiation, thereby showing a functional role for Mp1 both in normal differentiation of ES cells and in germ cell cancer.
PMCID: PMC3244037  PMID: 22143885
9.  Application of the New Classification on Patients with a Disorder of Sex Development in Indonesia 
Disorder of sex development (DSD) patients in Indonesia most often do not receive a proper diagnostic evaluation and treatment. This study intended to categorize 88 Indonesian patients in accordance with the new consensus DSD algorithm. Diagnostic evaluation including clinical, hormonal, genetic, imaging, surgical, and histological parameters was performed. Fifty-three patients were raised as males, and 34 as females. Of 22 patients with 46, XX DSD, 15 had congenital adrenal hyperplasia, while in one patient, an ovarian Leydig cell tumor was found. In all 58 46, XY DSD patients, 29 were suspected of a disorder of androgen action (12 with an androgen receptor mutation), and in 9, gonadal dysgenesis was found and, in 20, severe hypospadias e.c.i. Implementation of the current consensus statement in a resource-poor environment is very difficult. The aim of the diagnostic workup in developing countries should be to end up with an evidence-based diagnosis. This is essential to improve treatment and thereby to improve the patients' quality of life.
PMCID: PMC3255103  PMID: 22253624
10.  The Y-Encoded TSPY Protein: a Significant Marker Potentially Plays a Role in the Pathogenesis of Testicular Germ Cell Tumors 
Human pathology  2007;38(10):1470-1481.
The testis-specific protein Y-encoded (TSPY) gene is the putative gene for the gonadoblastoma locus on the Y chromosome (GBY) that predisposes dysgenetic gonads of intersex patients to gonadoblastoma development. TSPY is expressed at high levels in gonadoblastoma tissues, supporting its possible oncogenic function in this type of germ cell tumors. To explore the possibility that this Y chromosome gene is also involved in pathogenesis of the more common testicular germ cell tumors (TGCTs), we have conducted various expression studies using immunohistochemistry, Western blotting and RT-PCR analysis on 171 cases of TGCTs and selected normal testis controls. Our results demonstrated that TSPY protein is abundantly expressed in the precursor, carcinoma-in-situ or intratubular germ cell neoplasia unclassified (CIS/ITGCNU), and seminoma, but only minimally or not expressed in various types of nonseminomas. TSPY co-expresses with established germ cell tumor markers, such as PLAP, c-KIT, OCT4 and proliferative markers, such as Ki-67 and cyclin B1, in the same tumor cells at both RNA and protein levels. Ectopic TSPY expression in cultured cells up regulates pro-growth genes, including those at chromosome 12p13, frequently gained/amplified in TGCTs. Our results suggest that TSPY, in combination with other markers, could be an important marker for diagnosis and subclassification of TGCTs and support its role in the pathogenesis of both gonadoblastoma and TGCTs.
PMCID: PMC2744854  PMID: 17521702
gonadoblastoma locus; Y chromosome; TSPY; diagnostic marker; 12p genes; testicular germ cell tumors
11.  Resistance to Platinum-Containing Chemotherapy in Testicular Germ Cell Tumors Is Associated with Downregulation of the Protein Kinase SRPK11 
Neoplasia (New York, N.Y.)  2004;6(4):297-301.
Male germ cell tumors (GCTs) are extremely sensitive to platinum-containing chemotherapy, with only 10% of patients showing therapy resistance. However, the biological basis of the high curability of disseminated GCTs by chemotherapy is still unknown. Recently, we demonstrated that the mammalian serine/arginine-rich protein-specific kinase 1 (SRPK1) is a cisplatin-sensitive gene, inactivation of which leads to cisplatin resistance. Because, in mammalians, the expression of SRPK1 is preferentially high in testicular tissues, cisplatin responsiveness of male GCTs might be associated with SRPK1 levels. In the present study, we monitored SRPK1 protein expression in a unique series of nonseminomatous GCTs by immunohistochemistry. Randomly selected GCTs (n = 70) and tumors from patients responding to standard chemotherapy (n = 20) generally showed strong SRPK1 staining. In contrast, expression in refractory GCTs (n = 20) as well as in GCTs from poor-prognosis patients responding to high-dose chemotherapy only (n = 11) was significantly lower (two-sided Wilcoxon rank sum test: P < .001). In conclusion, our data suggest that SRPK1 expression might be an important prognostic indicator for the chemoresponsiveness of nonseminomatous GCTs.
PMCID: PMC1502111  PMID: 15256051
Chemotherapy resistance; germ cell tumors; chemotherapy sensitivity; protein kinase SRPK1; immunohistochemistry

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