http://repub.eur.nl/res/aut/363/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/38529/ 2012-11-27T00:00:00Z Background: Testicular Germ Cell Tumours (TGCT) are the most frequently occurring malignancy in males from 15-45 years of age. They are derived from germ cells unable to undergo physiological maturation, although the genetic basis for this is poorly understood. A recent report showed that mutations in the RNase IIIb domain of DICER1, a micro-RNA (miRNA) processing enzyme, are common in non-epithelial ovarian cancers. DICER1 mutations were found in 60% of Sertoli-Leydig cell tumours, clustering in four codons encoding metal-binding sites. Additional analysis of 14 TGCT DNA samples identified one case that also contained a mutation at one of these sites. Findings. A number of previous studies have shown that DICER1 mutations are found in <1% of most cancers. To provide a more accurate estimate of the frequency of such mutations in TGCTs, we have analysed 96 TGCT samples using high resolution melting curve analysis for sequence variants in these four codons. Although we did not detect any mutations in any of these sites, we did identify a novel mutation (c.1725 R>Q) within the RNase IIIb domain in one TGCT sample, which was predicted to disturb DICER1 function. Conclusion: Overall our findings suggest a mutation frequency in TGCTs of ∼1%. We conclude therefore that hot-spot mutations, frequently seen in Sertoli-Leydig cell tumours, are not common in TGCTs. http://repub.eur.nl/res/pub/38248/ 2012-02-03T00:00:00Z 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. Copyright http://repub.eur.nl/res/pub/30598/ 2011-11-30T00:00:00Z The aim of the work described in this thesis is to improve the understanding of the pathobiology of testicular cancer (type II Germ Cell Tumors) to create possibilities for optimalization of diagnosis for this type of malignancy in routine pathology laboratories. The different studies presented here show valuable additional information on the microscopic diagnostics in daily practice. This enables proper and complete diagnosis of this relative rare variant of cancer ensuring the best possible treatment for the patient. A number of novel sensitive and specific immunohistochemical markers are presented, of value for (early) diagnostics. In addition an enzymhistochemical method is presented informative to detect the presence of (pre-)malignant cells during a surgical intervention. Based on these data, a decision can be made regarding a testis sparing procedure. The studies included in this thesis are part of a diagnostically decision tree in which immuno- and enzymhistochemistry plays an important role. General Testicular cancer is a relative rare variant of cancer with raising incidence in the Western population, in contrast to the rest of the world. Within the Netherlands, about 750 new patients are diagnosed each year. In particular this affects young males between the age of 15 – 45 years The current treatment possibilities is successful in approximately 95% of the patients. It consists of removing of the affected testicle (radical orchiectomy) and possibly additional treatment using irradiation and/or chemotherapy. In spite of the excellent prognosis, patients often will suffer from long-term side effects of the treatment like fatigue, disorders of the metabolism, heart diseases, neurotoxicity and infertility. A small group of patients (5%) will shows an insensitivity of the cancer treatment given, and will possibly die of the disease. An early and accurate diagnosis can prevent under- or overtreatment, ensure an optimal balance between the pro and conts of the treatment. This will have significant impact on their quality of life, for both short- and long term. Specific The cell of origin from which testicular cancer arises is already present during the first months of pregnancy (embryogenesis), as demonstrated in Chapter 3. It is a primitive germ cell formed after the first month after fertilization, which provide spermatozoa after puberty by spermatogenesis. In case this primitive germ cell does not maturate completely and keeps the properties of the embryonal cell. This cell can become carcinoma in situ (CIS), being the progenitor of testicular cancer. These CIS cell is located in the testis on the same place as the spermatogonium, below the Sertoli cells on the basal lamina. These cells start to CHAPTER 14 multiply in an uncontrolled manner after puberty and finally they can develop cancer. In chapter 4 the totipotent character of this cancer is described for the first time in literature, shown by the presence of new formed germ cells. In other words testicular cancer is the only real totipotent cancer that can give rise to different germ lines the same as in embryogenesis. The application of a unique panel of immunological markers permitted this finding. In chapter 5 the value of the presence of OCT3/4 in CIS, seminoma and embryonal carcinoma is for the first time described. This study was initiated by the presence of this marker in embryonal cells and germ cells of the mouse. The diagnostic significance of this extremely sensitive and specific marker is huge as described in chapter 6. Diagnostics of testicular germ cell tumors requires in most cases surgical removed tissue. In chapter 7 a new method is described that offers the possibilities to detect OCT3/4 positive cells in semen. This method introduces the possibility to screen men with a higher risk for developing testicular germ cell tumor for CIS cells without surgical intervention. The robustness of this detection method for OCT3/4, even in sub optimal material, is illustrated in chapter 8. The malignant germ cells in the here described patient where only detectable by the presence of OCT3/4. The two groups of testicular cancer: seminomatous and nonseminomatous tumors, differs in sensitivity for irradiation and chemotherapy. Therefore differentiation of these two groups is of great importance for an optimal treatment. The nonseminomas can be subdivided in different groups of which embryonal carcinoma is the stem cell population. In chapter 9 detection of two SOX proteins (SOX2 and SOX17) is shown to be informative. Seminoma cells show expression of OCT3/4 and SOX17, embryonal carcinoma cells of OCT3/4 and SOX2. The other subgroups of the nonseminomas (yolk sac tumor, choriocarcinoma and teratoma) show a more heterogeneous expression. A chance of overdiagnosis of CIS in the testicle based on the presence of OCT3/4 is risk in young children, especially in the first year after birth, a result of a possible delay in maturation of the normal developing germ cells. This is Important because maturation delay is common also in cases of an increased risk for the development of testicular cancer, for instance an undescended testicle. In chapter 10 it is demonstrated how to distinguish a germ cell with maturation delay from a (pre-) malignant germ cell (CIS). Because SCF is only present in the direct neighbourhood of (pre-)malignant germ cells. This finding has an additional value in case of possible maturation delay when OCT3/4 positive cells are present. There is also a significant value for this finding concerning that specific DNA variants related to this gene, associated with an increased risk for development of testicular cancer in the entire population. SUMMARY/SAMENVATTING Organ sparing surgery may be preferred for different reasons, even so in cases of testicular cancer. Prevention of a second surgical intervention requires proper histopathological examination of frozen sections. In testis cancer this is mainly based on the detection of the presence of CIS. Unfortunately morphologic analysis of frozen material is not that easy, therefore formalin fixed tissue is needed which excludes often a single operation procedure. In chapter 11 a direct enzymhistochemical method is described which makes it possible to detect the presence of these CIS cells in a few minutes on frozen tissue. By using this direct alkaline phosphatase (dAP) technique it is possible to have certainty about the presence of CIS cells in the available tissue. Based on these results a decision is possible about performing a testis sparing operation. Chapter 12 describes the low DNA methylation status of both CIS cells and embryonal germ cells in their natural environment. This is performed by the use of immunohistochemistry. Interesting is the possible connection between cancer cells which are sensitive to cisplatin and the status of methylation. DNA of the resistant tumors seems to be hypermethylated compared to sensitive tumors. Analysis of cultured cell lines indicates c-FLIP gene as a potential candidate. This gene regulates the sensitivity of cells for a programmed cell death (apoptosis). Conclusions The studies presented give possibilities to perform optimal diagnostics of testicular cancer, eventually in certain circumstances, even in a non-invasive set up. These findings demonstrate that understanding of the normal development of germ cells, especially during embryogenesis, is of importance to carry out these kind of studies. The results give a solid base for implementation in a diagnostic set up and for further translational research. http://repub.eur.nl/res/pub/30996/ 2011-09-06T00:00:00Z Background: OCT3/4 (POU5F1) is an established diagnostic immunohistochemical marker for specific histological variants of human malignant germ cell tumours (GCTs), including the seminomatous types and the stem cell component of non-seminomas, known as embryonal carcinoma. OCT3/4 is crucial for the regulation of pluripotency and the self-renewal of normal embryonic stem-and germ cells. Detection of expression of this transcription factor is complicated by the existence of multiple pseudogenes and isoforms. Various claims have been made about OCT3/4 expression in non-GCTs, possibly related to using nonspecific detection methods. False-positive findings undermine the applicability of OCT3/4 as a specific diagnostic tool in a clinical setting. In addition, false-positive findings could result in misinterpretation of pluripotency regulation in solid somatic cancers and their stem cells. Of the three identified isoforms-OCT4A, OCT4B and OCT4B1-only OCT4A proved to regulate pluripotency. Up until now, no convincing nuclear OCT4A protein expression has been shown in somatic cancers or tissues. Methods: This study investigates expression of the various OCT3/4 isoforms in GCTs (both differentiated and undifferentiated) and somatic (non-germ cell) cancers, including representative cell lines and xenografts. Results: Using specific methods, OCT4A and OCT4B1 are shown to be preferentially expressed in undifferentiated GCTs. The OCT4B variant shows no difference in expression between GCTs (either differentiated or undifferentiated) and somatic cancers. In spite of the presence of OCT4A mRNA in somatic cancer-derived cell lines, no OCT3/4 protein is detected. Significant positive correlations between all isoforms of OCT3/4 were identified in both tumours with and without a known stem cell component, possibly indicating synergistic roles of these isoforms. Conclusion: This study confirms that OCT4A protein only appears in seminomatous GCTs, embryonal carcinoma and representative cell lines. Furthermore, it emphasises that in order to correctly assess the presence of functional OCT3/4, both isoform specific mRNA and protein detection are required. http://repub.eur.nl/res/pub/31238/ 2011-08-01T00:00:00Z Human type II germ cell tumours (GCTs) originate from an embryonic germ cell, either as a primordial germ cell or gonocyte. This start determines the biological as well as clinical characteristics of this type of cancer, amongst others their totipotency as well as their overall (exceptional) sensitivity to DNA damaging agents. The histology of the precursor lesion, either carcinoma in situ or gonadoblastoma, depends on the level of testicularization (i.e. testis formation) of the gonad. The impact of either intrinsic (genetic) - and environmental factors involved in the pathogenesis is demonstrated by disorders of sex development as well as testicular dysgenesis syndrome as risk factors, including cryptorchidism, hypospadias and disturbed fertility as parameters. This knowledge allows identification of individuals at risk for development of this type of cancer, being a population of interest for screening. Factors known to regulate pluripotency during embryogenesis are proven to be of diagnostic value for type II GCTs, including OCT3/4, even applicable for non-invasive screening. In addition, presence of stem cell factor, also known as KITLG, allows distinction between delayed matured germ cells and the earliest stages of malignant transformation. This is of special interest because of the identified association between development of type II GCTs of the testis and a limited number of single nucleotide polymorphisms, including some likely related to KITL. Transition from the precursor lesion to an invasive cancer is associated with gain of the short arm of chromosome 12, in which multiple genes might be involved, including KRAS2 and possibly NANOG (pseudogenes). While most precursor lesions will progress to an invasive cancer, only a limited number of cancers will develop treatment resistance. Putative explanatory mechanisms are identified, including presence of microsatellite instability, BRAF mutations, apoptosis suppression and p21 sub-cellular localization. It remains to be investigated how these different pathways integrate to each other and how informative they are at the patient-individual level. Further understanding will allow development of more targeted treatment, which will benefit quality of life of these young cancer patients. © 2011 The Authors. International Journal of Andrology http://repub.eur.nl/res/pub/31248/ 2011-08-01T00:00:00Z Aspects of the biopsy of the testis from the pathologist's point of view are discussed. Direct enzyme-histochemical staining for alkaline phosphatase (dAP) on frozen sections of biopsies taken during operation is a useful diagnostic tool to aid surgeons in testis-sparing surgery. Biopsy of the contralateral testis for the diagnosis of carcinoma in situ (CIS) in patients with a testicular germ cell tumour is not standard of care in most countries because of the high rate of negative biopsies. Based on risk factors for germ cell tumours, i.p. microlithiasis, a patient population is defined in which the rate of CIS in the contralateral biopsy is about 25%. It is reiterated that the diagnosis of CIS in testicular biopsies requires expertise, and should not be carried out without immunohistochemistry for markers for CIS. As OCT3/4 is increasingly used as marker, it is important to be aware that it may be false-negative in biopsies fixed in Bouin's or Stieve's fixative. Preliminary results are presented on a series of biopsies from cryptorchid testes in infants and children allowing the definition of morphological and immunohistochemical criteria for delayed maturation of gonocytes and pre-CIS. © 2011 The Authors. International Journal of Andrology http://repub.eur.nl/res/pub/31287/ 2011-08-01T00:00:00Z OCT3/4, NANOG, SOX2 and, most recently, LIN28 have been identified as key regulators of pluripotency in mammalian embryonic and induced stem cells, and are proven to be crucial for generation of the mouse germ-cell lineage. These factors are a hallmark of certain histological types of germ-cell tumours (GCTs). Here, we report novel information on the temporal and spatial expression pattern of LIN28 during normal human male germ-cell development as well as various types of GCTs. To investigate LIN28 expression, immunohistochemical analyses and quantitative proximity ligation assay-based TaqMan protein assays were applied on snap-frozen and formalin-fixed, paraffin-embedded samples as well as representative cell lines. LIN28 was found in primordial germ cells, gonocytes and pre-spermatogonia, in contrast to OCT3/4 and NANOG, which were found only in the first two stages. LIN28 was also found in all precursor lesions (carcinoma in situ and gonadoblastoma) of type II GCTs, as well as the invasive components seminoma and the non-seminomatous elements embryonal carcinoma and yolk sac tumour. Choriocarcinoma showed a heterogeneous pattern, while teratomas and spermatocytic seminomas (type III GCTs) were negative. This expression pattern suggests that LIN28 is associated with malignant behaviour of type II GCTs. Cell line experiments involving siRNA knockdown of LIN28, OCT3/4 and SOX2 showed that LIN28 plays a role in the maintenance of the undifferentiated state of both seminoma and embryonal carcinoma, closely linked to, and likely upstream of OCT3/4 and NANOG. In conclusion, LIN28 regulates the differentiation status of seminoma and embryonal carcinoma and is likely to play a related role in normal human germ-cell development. © 2011 The Authors. International Journal of Andrology http://repub.eur.nl/res/pub/33379/ 2011-07-01T00:00:00Z Context: Gonadectomy is avoided whenever possible in boys with 45,X/46,XY. However, no clinical markers are currently available to guide clinicians in predicting gonadal tumor risk or hormone production. Objective: The objective of the study was to test the hypothesis that gonadal histology and risk for development of a malignant germ cell tumor are reflected by the clinical presentation of a 45,X/46,XY individual. Design: The design of the study was the correlation of clinical data [external masculinization score (EMS), pubertal outcome] with pathology data (gonadal phenotype, tumor risk). Setting: This was a multicenter study involving two multidisciplinary disorder of sex development teams. Patients: Patients included genetically proven 45,X/46,XY (and variants) cases, ofwhomat leastone gonadal biopsy or gonadectomy specimen was available, together with clinical details. Interventions: Patients (n = 48) were divided into three groups, based on the EMS. Gonadal histology and tumor risk were assessedonparaffin-embedded samples (n = 87) by morphology and immunohistochemistry on the basis of established criteria. Main Outcome Measures: Gonadal differentiation and tumor risk in the three clinical groups were measured. Clinical outcome in patients with at least one preserved gonad was also measured. Results: Tumor risk in the three groups was significantly related to the gonadal differentiation pattern (P < 0.001). In boys, hormone production was sufficient and was not predicted by the EMS. Conclusions: The EMS reflects gonadal differentiation and tumor risk in patients with 45,X/46,XY. In boys, testosterone production is often sufficient, but strict follow-up is warranted because of malignancy risk, which appears inversely related to EMS. In girls, tumor risk is limited but gonads are not functional, makinggonadectomythemostreasonable option. Copyright http://repub.eur.nl/res/pub/23886/ 2011-02-01T00:00:00Z Aims: Testis-sparing surgery might benefit quality of life, but can only be applied with histological examination for the presence of invasive germ cell tumour components, and the precursor carcinoma in situ (CIS). Currently, diagnosis is based on paraffin-embedded tissue, therefore a delay in further surgery is mainly unavoidable. The aim was to develop an intraoperative assessment technique using alkaline phosphatase as a marker. Methods and results: A total of 4093 snap-frozen samples and matched paraffin-embedded tissue of 1500 patients were included. Besides standard haematoxylin and eosin (H&E) staining, the direct enzymatic alkaline phosphatase reactivity (dAP) test (duration 15min) was applied on frozen sections, while H&E and immunohistochemistry for detection of OCT3/4, α-fetoprotein, human chorionic gonadotrophin (hCG) and cytokeratin was performed on the paraffin-embedded slides. Endothelial cells served as control for the dAP test. Positive staining was found in all CIS (n=965), seminoma (n=1035) and embryonal carcinoma (n=584), either intratubular, microinvasive or invasive. Differentiated non-seminomas (n=1238) showed variable staining. No staining was identified in spermatocytic seminomas (n=5), testicular lymphomas (n=42), testicular rhabdomyosarcomas (n=7), Leydig cell tumours (n=31), Sertoli-cell-only nodules (n=4), (epi) dermoid cyst (n=16), normal testicular parenchyma (n=116), testicular torsion (n=32) and inflammation of the epididymis (n=19). The dAP test results matched H&E-stained parallel sections, as well paraffin-embedded tissue analysis, including immunohistochemistry. Conclusions: The dAP test is an informative, reproducible and easy tool to diagnose CIS, (intratubular and microinvasive) seminoma and embryonal carcinoma on frozen tissue sections, being of great value in the context of sparing surgery. http://repub.eur.nl/res/pub/28586/ 2010-04-01T00:00:00Z Disorders of sex development (DSD), previously referred to as intersex, has been recognised as one of the main risk factors for development of type II germ cell tumours (GCTs), that is, seminomas/dysgerminomas and non-seminomas (e.g., embryonal carcinoma, yolk sac tumour, choriocarcinoma and teratoma). Within the testis, this type of cancer is the most frequent malignancy in adolescent and young adult Caucasian males. Although these males are not known to have dysgenetic gonads, the similarities in the resulting tumours suggest a common aetiological mechanism(s), -genetically, environmentally or a combination of both. Within the group of DSD patients, being in fact congenital conditions, the risk of malignant transformation of germ cells is highly heterogeneous, depending on a number of parameters, some of which have only recently been identified. Understanding of these recent insights will stimulate further research, with the final aim to develop an informative clinical decision tree for DSD patients, which includes optimal (early) diagnosis without overtreatment, such as prophylactic gonadectomy in the case of a low tumour risk. http://repub.eur.nl/res/pub/24772/ 2009-12-01T00:00:00Z Carcinoma in situ (CIS) is the common precursor of all type II testicular germ cell tumors (TGCTs), i.e. seminomas and non-seminomas, which can be diagnosed using a surgical biopsy. The objective of this study was to investigate the additional value of immunohistochemistry for the diagnosis of CIS in assessing testicular biopsies taken in the context of infertility. A series of 21 infertile patients were retrieved from the Dutch pathological database (PALGA), being diagnosed with an invasive TGCT, while a matched previously obtained testicular biopsy was diagnosed as non-malignant. From 20 patients, both the invasive tumors as well as the biopsies were revised using morphology and immunohistochemistry for OCT3/4, placental-like alkaline phosphatase and c-KIT, all known established markers for CIS. The presence of CIS or invasive malignancies was scored. There are no interventions. Morphological criteria alone allowed an experienced pathologist in TGCTs to diagnose CIS in five and an invasive tumor in two cases (total n = 7, 35%). Application of immunohistochemistry resulted in the identification of an additional four cases of CIS (total n = 11, 55%, additional value of 20%). The initial correct diagnosis of CIS could have prevented a second gonadectomy in four patients (20%). This study, for the first time, really shows that time of progression from CIS to seminoma is longer than to non-seminoma. Our study demonstrates that immunohistochemistry should be performed for the diagnosis of CIS of the testis on single biopsies obtained because of infertility, resulting in an extra diagnostic yield of at least 20%. Application of this protocol will allow early diagnosis, and therefore prevent any adverse anti-cancer treatment sequelae including gonadectomy, and requiring life long androgen supplementation in some patients. http://repub.eur.nl/res/pub/24551/ 2009-06-12T00:00:00Z Patients with disorders of sex development (DSD), especially those with gonadal dysgenesis and hypovirilization, are at risk of developing the so-called type II germ cell tumors (GCTs). Both carcinoma in situ and gonadoblastoma (GB) can be the precursor lesion, resulting in a seminomatous or non-seminomatous invasive cancer. SRY mutations residing in the HMG domain are found in 10-15% of 46,XY gonadal dysgenesis cases. This domain contains two nuclear localization signals (NLSs). In this study, we report a unique case of a phenotypical normal woman, diagnosed as a patient with 46,XY gonadal dysgenesis, with an NLS missense mutation, on the basis of the histological diagnosis of a unilateral GB. The normal role of SRY in gonadal development is the upregulation of SOX9 expression. The premalignant lesion of the initially removed gonad was positive for OCT3/4, TSPY and stem cell factor in germ cells, and for FOXL2 in the stromal component (ie, granulosa cells), but not for SOX9. On the basis of these findings, prophylactical gonadectomy of the other gonad was performed, also showing a GB lesion positive for both FOXL2 (ovary) and SOX9 (testis). The identified W70L mutation in the SRY gene resulted in a 50% reduction in the nuclear accumulation of the mutant protein compared with wild type. This likely explains the diminished SOX9 expression, and therefore the lack of proper Sertoli cell differentiation during development. This case shows the value of the proper diagnosis of human GCTs in identification of patients with DSD, which allows subsequent early diagnosis and prevention of the development of an invasive cancer, likely to be treated by chemotherapy at young age. http://repub.eur.nl/res/pub/15949/ 2008-09-01T00:00:00Z Carcinoma in situ (CIS) of the testis is the pre-invasive stage of type II testicular germ cell tumours (TGCTs) of adolescents and adults. These tumours are the most frequently diagnosed cancer in Caucasian adolescents and young adults. In dysgenetic gonads, the precursor of type II GCTs can be either CIS or a lesion known as gonadoblastoma (GB). CIS/GB originates from a primordial germ cell (PGC)/gonocyte, ie an embryonic cell. CIS can be cured by local low-dose irradiation, with limited side effects on hormonal function. Therefore, strategies for early diagnosis of CIS are essential. Various markers are informative to diagnose CIS in adult testis by immunohistochemistry, including c-KIT, PLAP, AP-2γ, NANOG, and POU5F1 (OCT3/4). OCT3/4 is the most informative and consistent in presence and expression level, resulting in intense nuclear staining. In the case of maturational delay of germ cells, frequently present in gonads of individuals at risk for type II (T)GCTs, use of these markers can result in overdiagnosis of malignant germ cells. This demonstrates the need for a more specific diagnostic marker to distinguish malignant germ cells from germ cells showing maturation delay. Here we report the novel finding that immunohistochemical detection of stem cell factor (SCF), the c-KIT ligand, is informative in this context. This was demonstrated in over 400 cases of normal (fetal, neonatal, infantile, and adult) and pathological gonads, as well as TGCT-derived cell lines, specifically in cases of CIS and GB. Both membrane-bound and soluble SCF were expressed, suggestive of an autocrine loop. SCF immunohistochemistry can be a valuable diagnostic tool, in addition to OCT3/4, to screen for precursor lesions of TGCTs, especially in patients with germ cell maturation delay. http://repub.eur.nl/res/pub/29674/ 2008-07-01T00:00:00Z Objective: Carcinoma in situ (CIS) is accepted as the precursor of the germ cell tumors of the adult testis. CIS cells are located within the seminiferous tubules and can be exfoliated into semen. We performed a study to detect CIS cells in semen using the highly specific immunohistochemical marker OCT3/4, potentially a method for noninvasive diagnosis. Material and methods: In 2006, 41 men at risk for CIS of the testis were found eligible for this study. Indications for inclusions were a suspicious lesion on scrotal ultrasound investigation (n = 14), patients on surveillance after a history of a testicular tumor (n = 14), and 13 patients with bilateral testicular microlithiasis (TM). Results: Three of the 13 men (23%) who underwent testicular biopsies for bilateral TM were histologically diagnosed with CIS (two bilateral), and their semen showed OCT3/4-positive cells in all cases. Twelve of the 14 patients (86%) with a solid mass were diagnosed with a TGCT with adjacent CIS in the parenchyma, and in 9 cases (75%) OCT3/4-positive cells were present in the semen. No OCT3/4-positive cells were found in patients with biopsies who did not show any evidence of malignancy. Conclusion: This study demonstrates that OCT3/4-positive cells can be found in semen from the majority of patients with CIS. The observations indicate that there is probably a time window in which the CIS cells are exfoliated, which gives an opportunity for early detection of CIS cells in semen of men at risk for TGCT. http://repub.eur.nl/res/pub/28917/ 2008-05-01T00:00:00Z The transcription factors SOX9 and FOXL2 are required for male and female mammalian gonadal development. We have used specific antibodies to investigate the role of these key proteins in disorders of sex development (DSD), specifically inter-sex states. In normal gonads, SOX9 was found to be restricted to the presence of (pre-)Sertoli cells, while FOXL2 was found in granulosa cells, and in stromal cells interpreted as early ovarian stroma. Both proteins were found within a single patient, when testicular and ovarian development was present; and within the same gonad, when both differentiation lineages were identified, as in ovotesticular DSD (ie hermaphrodite). Especially SOX9 was informative to support the presence of early testicular development (ie seminiferous tubules), expected based on morphological criteria only. In a limited number of DSD cases, FOXL2 was found within reasonably well-developed seminiferous tubules, but double staining demonstrated that it was never strongly co-expressed with SOX9 in the same cell. All seminiferous tubules containing carcinoma in situ (CIS), the malignant counterpart of a primordial germ cell, ie the precursor of type II germ cell tumours of the testis, seminomas and non-seminomas, showed the presence of SOX9 and not FOXL2. In contrast, gonadoblastomas (GBs), the precursor of the same type of cancer, in a dysgenetic gonad, showed expression of FOXL2 and no, or only very low, SOX9 expression. These findings indicate that gonadal differentiation, ie testicular or ovarian, determines the morphology of the precursor of type II germ cell tumours, CIS or GB, respectively. We show that in DSD patients, the formation of either ovarian or/and testicular development can be visualized using FOXL2 and SOX9 expression, respectively. In addition, it initiates a novel way to study the role of the supportive cells in the development of either CIS or GB. Copyright http://repub.eur.nl/res/pub/30047/ 2008-03-01T00:00:00Z Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs. http://repub.eur.nl/res/pub/30057/ 2008-01-01T00:00:00Z 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. http://repub.eur.nl/res/pub/30120/ 2008-01-01T00:00:00Z A 40-year-old woman presented with blurred vision and diplopia, followed by slowly progressive left-sided motor and sensory disturbances. She also suffered from memory loss and had mild spatial and temporal disorientation. A T2-weighted MRI showed a large area of high signal intensity in the periventricular white matter of the right more than the left occipital region and the corpus callosum, without enhancement on T1-weighted images after gadolinium administration and without mass effect. A stereotacticbiopsy of the intracerebral lesion showed blast-like neoplastic cells within a mononuclear infiltrate. No diagnosis could be made based on morphology and immunohistochemistry using a large series of markers. However, based on positive OCT3/4 nuclear staining, the tumor was diagnosed as a germinoma (seminoma of the brain). The patient was treated accordingly and her condition improved, although focal deficits remained. http://repub.eur.nl/res/pub/35122/ 2007-11-01T00:00:00Z Testicular germ cell tumours (GCTs) of adolescents and adults can be subdivided into seminomas (referred to as dysgerminomas of the ovary) and non-seminomas, all referred to as type II GCTs. They originate from carcinoma in situ (CIS), being the malignant counterparts of primordial germ cells (PGCs)/gonocytes. The invasive components mimic embryogenesis, including the stem cell component embryonal carcinoma (EC), the somatic lineage teratoma (TE), and the extra-embryonic tissues yolk sac tumour (YST) and choriocarcinoma (CH). The other type is the so-called spermatocytic seminomas (SS, type III GCT), composed of neoplastic primary spermatocytes. We reported previously that the miRNAs hsa-miR 371-373 cluster is involved in overruling cellular senescence induced by oncogenic stress, allowing cells to become malignant. Here we report the first high-throughput screen of 156 microRNAs in a series of type II and III GCTs (n = 69, in duplicate) using a quantitative PCR-based approach. After normalization to allow intersample analysis, the technical replicates clustered together, and the previous hsa-miRNA 371-373 cluster finding was confirmed. Unsupervised cluster analysis demonstrated that the cell lines are different from the in vivo samples. The in vivo samples, both normal and malignant, clustered predominantly based on their maturation status. This parallels normal embryogenesis, rather than chromosomal anomalies in the tumours. miRNAs within a single cluster showed a similar expression pattern, implying common regulatory mechanisms. Normal testicular tissue expressed most discriminating miRNAs at a higher level than SE and SS. Moreover, differentiated non-seminomas showed overexpression of discriminating miRNAs. These results support the model that miRNAs are involved in regulating differentiation of stem cells, retained in GCTs. Copyright http://repub.eur.nl/res/pub/35750/ 2007-08-01T00:00:00Z The JKT-1 cell line has been used in multiple independent studies as a representative model of human testicular seminoma. However, no cell line for this specific tumour type has been independently confirmed previously; and therefore, the seminomatous origin of JKT-1 must be proven. The genetic constitution of the JKT-1 cells was determined using flow cytometry and spectral karyotyping, as well as array comparative genomic hybridization and fluorescent in situ hybridization. Marker profiling, predominantly based on differentially expressed proteins during normal germ cell development, was performed by immunohistochemistry and Western blot analyses. Moreover, genome wide affymetrix mRNA expression and profiling of 157 microRNAs was performed, and the status of genomic imprinting was determined. A germ cell origin of the JKT-1 cells was in line with genomic imprinting status and marker profile (including positive staining for several cancer-testis antigens). However, the supposed primary tumour, from which the cell line was derived, being indeed a classical seminoma, was molecularly proven not to be the origin of the cell line. The characteristic chromosomal anomalies of seminoma, e.g. gain of the short arm of chromosome 12, as well as the informative marker profile (positive staining for OCT3/4, NANOG, among others) were absent in the various JKT-1 cell lines investigated, irrespective of where the cells were cultured. All results indicate that the JKT-1 cell line is not representative of human seminoma. Although it can originate from an early germ cell, a non-germ cell derivation cannot be excluded. http://repub.eur.nl/res/pub/36050/ 2007-08-01T00:00:00Z Objective: Gonadal karyotyping is considered a tool for increasing our knowledge of disturbed gonadal development in patients with gonadal dysgenesis and for estimating more accurately the risk for gonadoblastoma formation. The objective was to gain insight into the role of Y chromosome distribution in the histological heterogeneity of gonads of patients with gonadal dysgenesis. Design: Investigation of the possible relationship between peripheral blood karyotype, gonadal karyotype, morphological differentiation patterns of dysgenetic gonads and tumour formation. Patients: In total 22 gonadal samples from 19 patients with gonadal dysgenesis (45,X/46,XY and variants n = 14; 46,XY: n = 3; 46,XX: n = 2) were examined. Measurements: Morphological examination and immunohistochemical staining for testis specific protein, Y encoded (TSPY) and fluorescent and nonfluorescent in situ hybridization directly on gonadal tissue. Results: No correlation was observed between peripheral blood karyotype and gonadal karyotype or between gonadal karyotype and the corresponding differentiation pattern. A Y-containing cell line in Sertoli cells was encountered no more frequently than were other cell types. Conclusions: The distribution of the Y-containing cell line in peripheral blood is not a suitable indicator for predicting the histological differentiation pattern found in the gonads of patients with gonadal dysgenesis. The analysis of Y-containing cell lines in the gonads of such patients could be informative with regard to the specific characteristics of gonadal development in humans as compared to chimeric mouse models. Moreover, it is essential to understand the mechanisms underlying disturbed gonadogenesis in these patients. As the gonadal karyotype is not related to the encountered gonadal differentiation pattern, it does not allow prediction of the risk for gonadoblastoma formation. http://repub.eur.nl/res/pub/10100/ 2003-01-01T00:00:00Z PURPOSE: Germ cell tumors (GCTs) are highly sensitive to cisplatin-based chemotherapy. This feature is unexplained, as is the intrinsic chemotherapy resistance of mature teratomas and the resistant phenotype of a minority of refractory GCTs. Various cellular pathways may influence the efficacy of chemotherapy. Their impact has not been investigated in a comprehensive study of tumor samples from clinically defined subgroups of GCT patients. EXPERIMENTAL DESIGN: We investigated proteins involved in regulation of apoptosis (p53, BAX, BCL-2, and BCL-X(L)), cell cycle control [p21 and retinoblastoma protein (RB)], and drug export and inactivation [P-glycoprotein, multidrug resistance-associated protein (MRP) 1, MRP2, breast cancer resistance protein, lung resistance protein, metallothionein, and glutathione S-transferase pi] immunohistochemically in samples of unselected GCT patients (n = 20), patients with advanced metastatic disease in continuous remission after first-line chemotherapy (n = 12), and chemotherapy-refractory patients (n = 24). Mature teratoma components (n = 10) within tumor samples from all groups were analyzed separately. The apoptotic index was studied by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. RESULTS: Invasive GCTs of all groups showed a correlation between wild-type p53 and apoptotic index (r(s) = 0.66; P < 0.001). The levels of the antiapoptotic proteins BCL-2 and BCL-X(L) were generally low. p21 was hardly detectable and did not correlate with p53 (r(s) = 0.29; P = 0.07). No significant differences among the three patient groups were identified regarding any of the investigated parameters (all Ps were >0.08), even though only individual samples from chemotherapy-resistant cases showed a strong staining for MRP2 and GSTpi. In contrast to other components, mature teratomas showed an intense p21 and RB staining and were mostly positive for MRP2, lung resistance protein, and GSTpi. CONCLUSIONS: Our results indicate a multifactorial basis for the chemosensitivity of GCTs with lack of transporters for cisplatin, of antiapoptotic BCL-2 family members, of p21 induction by p53, and of RB and an intact apoptotic cascade downstream of p53. These findings suggest a preference for apoptosis over cell cycle arrest after up-regulation of p53. None of the examined parameters offers a general explanation for the chemotherapy-resistant phenotype of refractory tumors. The up-regulation of various factors interfering with chemotherapy efficacy and ability for a p21-induced cell cycle arrest may explain the intrinsic chemotherapy resistance of mature teratomas. http://repub.eur.nl/res/pub/10129/ 2003-01-01T00:00:00Z Human germ cell tumors (GCTs) may have variable histology and clinical behavior, depending on factors such as sex of the patient, age at clinical diagnosis, and anatomical site of the tumor. Some types of GCT, i.e., the seminomas/germinomas/dysgerminomas and embryonal carcinomas (the stem cell component of nonseminomas), have pluripotent potential, which is demonstrated by their capacity to differentiate into somatic and/or extraembryonic elements. Although embryonal carcinoma cells are intrinsically pluripotent, seminoma/germinoma/dysgerminoma cells, as well as their precursor carcinoma in situ/gonadoblastoma cells, have the phenotype of early germ cells that can be activated to pluripotency. The other types of GCT (teratomas and yolk sac tumors of infants and newborn, dermoid cyst of the ovary, and spermatocytic seminoma of elderly) are composed of (fully) differentiated tissues and lack the appearance of undifferentiated and pluripotent stem cells. OCT3/4, a transcription factor also known as OTF3 and POU5F1, is involved in regulation of pluripotency during normal development and is detectable in embryonic stem and germ cells. We analyzed the presence of POU5F1 in GCT and other tumor types using immunohistochemistry. The protein was consistently detected in carcinoma in situ/gonadoblastoma, seminomas/germinoma/dysgerminoma, and embryonal carcinoma but not in the various types of differentiated nonseminomas. Multitumor tissue microarray analysis covering >100 different tumor categories and 3600 individual cancers verified that POU5F1 expression is specific for particular subtypes of GCT of adults. No protein was observed in GCT of newborn and infants, spermatocytic seminomas, and the various tumors of nongerm cell origin. In addition, no difference in staining pattern was found in chemosensitive and chemoresistant GCT of adults. These results indicate preservation of the link between POU5F1 and pluripotency, as reported during normal development, after malignant transformation. Therefore, POU5F1 immunohistochemistry is an informative diagnostic tool for pluripotent GCT and offers new insights into the histological heterogeneity of this cancer. http://repub.eur.nl/res/pub/10242/ 2003-01-01T00:00:00Z Testicular germ-cell tumors (TGCTs) of adolescents and adults originate from intratubular germ cell neoplasia (ITGCN), which is composed of the malignant counterparts of embryonal germ cells. ITGCN cells are characterized, among others, by the presence of stem cell factor receptor c-KIT. Once established, ITGCN will always progress to invasiveness. Approximately 2.5-5% of patients with a TGCT will develop bilateral disease and require complete castration, resulting in infertility, a need for lifelong androgen replacement, and psychological stress. To date, the only way to predict a contralateral tumor is surgical biopsy of the contralateral testis to demonstrate ITGCN. We did a retrospective study of 224 unilateral and 61 proven bilateral TGCTs (from 46 patients, in three independently collected series in Europe) for the presence of activating c-KIT codon 816 mutations. A c-KIT codon 816 mutation was found in three unilateral TGCT (1.3%), and in 57 bilateral TGCTs (93%; P < 0.0001). In the two wild-type bilateral tumors for which ITGCN was available, the preinvasive cells contained the mutation. The mutations were somatic in origin and identical in both tumors. We conclude that somatic activating codon 816 c-KIT mutations are associated with development of bilateral TGCT. Detection of c-KIT codon 816 mutations in unilateral TGCT identifies patients at risk for bilateral disease. These patients may undergo tailored treatment to prevent the development of bilateral disease, with retention of testicular hormonal function. http://repub.eur.nl/res/pub/9877/ 2002-01-01T00:00:00Z Seminomas and nonseminomas represent the invasive stages of testicular (TGCTs) of adolescents and adults. Although TGCTs are characterized by extra copies of the short arm of chromosome 12, the genetic basis for gain of 12p in the pathogenesis of this cancer is not yet understood. We have demonstrated that gain of 12p is related to invasive growth and that amplification of specific 12p sequences, i.e., 12p11.2-p12.1, correlates with reduced apoptosis in the tumors. Here we show that three known genes map within the newly determined shortest region of overlap of amplification (SROA): DAD-R, SOX5, and EKI1. Whereas EKI1 maps close to the telomeric region of the SROA, DAD-R is the first gene at the centromeric region within the 12p amplicon. Although all three genes are amplified to the same level within the SROA, expression of DAD-R is significantly up-regulated in seminomas with the restricted 12p amplification compared with seminomas without this amplicon. DAD-R is also highly expressed in nonseminomas of various histologies and derived cell lines, both lacking such amplification. This finding is of particular interest because seminomas with the restricted 12p amplification and nonseminomas are manifested clinically in the third decade of life and show a low degree of apoptosis. In contrast, seminomas lacking a restricted 12p amplification, showing significantly lower levels of DAD-R with pronounced apoptosis, manifest clinically in the fourth decade of life. A low level of DAD-R expression is also observed in normal testicular parenchyma and in parenchyma containing the precursor cells of this cancer, i.e., carcinoma in situ. Therefore, elevated DAD-R expression in seminomas and nonseminomas correlates with invasive growth and a reduced level of apoptosis associated with an earlier clinical presentation. These data implicate DAD-R as a candidate gene responsible in part for the pathological effects resulting from gain of 12p sequences in TGCTs. In addition, our results also imply differences in expression regulation of DAD-R between seminomas and nonseminomas. http://repub.eur.nl/res/pub/9479/ 2000-01-01T00:00:00Z Human testicular germ-cell tumors of young adults (TGCTs), both seminomas and nonseminomas, are characterized by 12p overrepresentation, mostly as isochromosomes, of which the biological and clinical significance is still unclear. A limited number of TGCTs has been identified with an additional high-level amplification of a restricted region of 12p including the K-RAS proto-oncogene. Here we show that the incidence of these restricted 12p amplifications is approximately 8% in primary TGCTs. Within a single cell formation of i(12p) and restricted 12p amplification is mutually exclusive. The borders of the amplicons cluster in short regions, and the amplicon was never found in the adjacent carcinoma in situ cells. Seminomas with the restricted 12p amplification virtually lacked apoptosis and the tumor cells showed prolonged in vitro survival like seminoma cells with a mutated RAS gene. However, no differences in proliferation index between these different groups of seminomas were found. Although patients with a seminoma containing a homogeneous restricted 12p amplification presented at a significantly younger age than those lacking it, the presence of a restricted 12p amplification/RAS mutation did not predict the stage of the disease at clinical presentation and the treatment response of primary seminomas. In 55 primary and metastatic tumors from 44 different patients who failed cisplatinum-based chemotherapy, the restricted 12p amplification and RAS mutations had the same incidence as in the consecutive series of responding patients. These data support the model that gain of 12p in TGCTs is related to invasive growth. It allows tumor cells, in particular those showing characteristics of early germ cells (ie, the seminoma cells), to survive outside their specific microenvironment. Overexpression of certain genes on 12p probably inhibits apoptosis in these tumor cells. However, the copy numbers of the restricted amplification of 12p and K-RAS mutations do not predict response to therapy and survival of the patients.