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We report detailed functional analyses and genotype-phenotype correlations in 392 individuals carrying disease-causing variants in SCN8A, encoding the voltage-gated Na+ channel Nav1.6, with the aim of describing clinical phenotypes related to functional effects. Six different clinical subgroups were identified: Group 1, benign familial infantile epilepsy (n = 15, normal cognition, treatable seizures); Group 2, intermediate epilepsy (n = 33, mild intellectual disability, partially pharmaco-responsive); Group 3, developmental and epileptic encephalopathy (n = 177, severe intellectual disability, majority pharmaco-resistant); Group 4, generalized epilepsy (n = 20, mild to moderate intellectual disability, frequently with absence seizures); Group 5, unclassifiable epilepsy (n = 127); and Group 6, neurodevelopmental disorder without epilepsy (n = 20, mild to moderate intellectual disability). Those in Groups 1–3 presented with focal or multifocal seizures (median age of onset: 4 months) and focal epileptiform discharges, whereas the onset of seizures in patients with generalized epilepsy was later (median: 42 months) with generalized epileptiform discharges. We performed functional studies expressing missense variants in ND7/23 neuroblastoma cells and primary neuronal cultures using recombinant tetrodotoxin-insensitive human Nav1.6 channels and whole-cell patch-clamping. Two variants causing developmental and epileptic encephalopathy showed a strong gain-of-function (hyperpolarizing shift of steady-state activation, strongly increased neuronal firing rate) and one variant causing benign familial infantile epilepsy or intermediate epilepsy showed a mild gain-of-function (defective fast inactivation, less increased firing). In contrast, all three variants causing generalized epilepsy induced a loss-of-function (reduced current amplitudes, depolarizing shift of steady-state activation, reduced neuronal firing). Functional effects were known for 170 individuals. All 136 individuals carrying a functionally tested gain-of-function variant had either focal (n = 97, Groups 1–3) or unclassifiable (n = 39) epilepsy, whereas 34 individuals with a loss-of-function variant had either generalized (n = 14), no (n = 11) or unclassifiable (n = 6) epilepsy; only three had developmental and epileptic encephalopathy. Computational modelling in the gain-of-function group revealed a significant correlation between the severity of the electrophysiological and clinical phenotypes. Gain-of-function variant carriers responded significantly better to sodium channel blockers than to other anti-seizure medications, and the same applied for all individuals in Groups 1–3. In conclusion, our data reveal clear genotype-phenotype correlations between age at seizure onset, type of epilepsy and gain- or loss-of-function effects of SCN8A variants. Generalized epilepsy with absence seizures is the main epilepsy phenotype of loss-of-function variant carriers and the extent of the electrophysiological dysfunction of the gain-of-function variants is a main determinant of the severity of the clinical phenotype in focal epilepsies. Our pharmacological data indicate that sodium channel blockers present a treatment option in SCN8A-related focal epilepsy with onset in the first year of life.

Carcinoma in situ (CIS) is the common precursor of histologically heterogeneous testicular germ cell tumors (TGCTs), which in recent decades have markedly increased and now are the most common malignancy of young men. Using genome-wide gene expression profiling, we identified >200 genes highly expressed in testicular CIS, including many never reported in testicular neoplasms. Expression was further verified by semiquantitative reverse transcription-PCR and in situ hybridization. Among the highest expressed genes were NANOG and POU5F1, and reverse transcription-PCR revealed possible changes in their stoichiometry on progression into embryonic carcinoma. We compared the CIS expression profile with patterns reported in embryonic stem cells (ESCs), which revealed a substantial overlap that may be as high as 50%. We also demonstrated an over-representation of expressed genes in regions of 17q and 12, reported as unstable in cultured ESCs. The close similarity between CIS and ESCs explains the pluripotency of CIS. Moreover, the findings are consistent with an early prenatal origin of TGCTs and thus suggest that etiologic factors operating in utero are of primary importance for the incidence trends of TGCTs. Finally, some of the highly expressed genes identified in this study are promising candidates for new diagnostic markers for CIS and/or TGCTs.

Genes found up-regulated in testicular samples with CIS (fold change)

Supplementary Figure 2 from Embryonic Stem Cell-Like Features of Testicular Carcinoma in Situ Revealed by Genome-Wide Gene Expression Profiling

Supplementary Figure 1 from Embryonic Stem Cell-Like Features of Testicular Carcinoma in Situ Revealed by Genome-Wide Gene Expression Profiling

Background: Preterm infants have an increased risk of neurodevelopmental disorders. We established a direct quantitative comparison of the association between the degree of prematurity and three different neurodevelopmental disorders. Methods: In this cohort study, we combined data from 995,498 children in the Danish Medical Birth Register, from birth years 1997–2013, with information on cerebral palsy, epilepsy, and special educational needs. We estimated the gestational week-specific prevalence and risk for each of the disorders. Results: The risk ratio of cerebral palsy at gestational weeks 21–24, compared to term birth, was more than ten times higher than for the two other disorders. The prevalence of epilepsy and special educational needs declined almost parallel, with 9.2% (4.6%–13.5%) and 12.5% (11.2%–13.7%), respectively, per week of gestation toward term birth. Cerebral palsy did not decline similarly: from gestational weeks 21–24 until week 29 the prevalence declined insignificantly by 0.6% (−11.1%–11.0%) per week; whereas from week 29 until term, the prevalence declined markedly by 36.7% (25.9%–45.9%) per week. Conclusions: The prevalence and risk of cerebral palsy are affected differently by the degree of prematurity compared with epilepsy and special educational needs, possibly reflecting important differences in cerebral pathophysiology. Impact: For each week of gestation toward term birth, there was a clear log-linear decline in the prevalence of early childhood epilepsy and special educational needs.In contrast, the risk of cerebral palsy was high at the earliest gestational age, and the prevalence did not decline significantly until gestational week 29, from where it declined notably by nearly 40% for each week of gestation until term birth.Our results indicate important differences in the pathophysiological processes that associate preterm birth with these three neurodevelopmental disorders.

This review finds that, in children and adults with epilepsy, there are several treatment options. Multiple antiseizure medications are available and in case of drug-resistant epilepsy, a non-pharmacological approach is recommended, including epilepsy surgery, vagus nerve stimulation, or ketogenic diet treatment. The aim of the treatment is to avoid further seizures, but also to avoid negative cognitive, psychological, and social consequences of epilepsy.

The recurrent seizures of pediatric drug-resistant epilepsy (DRE) are known to impair brain development and can lead to a loss in cognitive functioning. Surgery is increasingly being used to treat children with DRE. This study investigates the pre- and postoperative cognitive function in a pediatric epilepsy surgery cohort as well as predictive determinants of change in intelligence quotient (IQ) following surgery.A consecutive series of 91 Danish children who underwent focal resective epilepsy surgery between January 1996 and December 2016 were included. All underwent preoperative cognitive evaluation and were reevaluated at 1-year and/or 2-year follow-up. Single-operated and multi-operated patients were examined separately.79 of 91 patients were single-operated. Single-operated patients received less anti-epileptic drugs (AED) and experienced a decrease in seizure frequency postoperatively, p0.001. IQ increased postoperatively (IQ change ± standard deviation: 3.3 ± 14.0), p0.05. High preoperative seizure frequency was a significant predictor for decreased IQ, p0.01. Multi-operated patients did not experience a reduction in AED treatment. Surgery and continued AED treatment did, however, result in significantly better seizure control, p0.01. IQ remained unchanged in multi-operated patients.Epilepsy surgery allowed for IQ gains in single-operated patients. Preoperative seizure frequency was a significant predictor of IQ change following surgery. Interactions between other, not included, possible predictors remain to be examined. Single-operated patients had the best cognitive outcome. The inclusion of a non-surgical control group is needed to assess the extent of the beneficial effects of surgery on cognitive ability.

Background: The use of homemade tube feeding formula has become increasingly popular for children requiring enteral nutrition. This project aimed to investigate nutrition and preparation of blenderized tube feeding in the field of children and adolescents with neurological impairment.Methods: A scoping review was performed using established methodologies. In January 2021, we searched PubMed, Embase, CINAHL Complete, the Cochrane Central Register of Controlled Trials, and gray literature to identify relevant articles.Major findings: Twenty-two papers were included describing the composition of food items, preparation procedures, and food safety. No randomized controlled trials and only a few prospective studies were included. A broad variety of food items from all food groups and many examples of recipes were presented. Most recipes provided 1.0 kcal/ml but tended to contain less energy and nutrients than expected, which could be due to preparation issues, such as sieving and the high viscosity of the blend. Preparation requires a commercial-grade household blender and diligence to ensure thorough household hygiene for adequate food safety.Conclusions: This review revealed practical experience in the nutrition and preparation aspects of blenderized tube feeding but minimal empirical evidence. Multiple examples of the composition of food items and preparation procedures for blenderized tube feeding were found, but uncertainty regarding the ideal composition or preparation was also exposed. The future of blenderized tube feeding would benefit from clinically tested recipes that include an evaluation of nutrients, viscosity, and microbial contamination, as well as the effect of the food's appearance and scent on the target group.

Aim: Children with dyskinetic cerebral palsy (CP) are often severely affected and effective treatment is difficult, due to different underlying disease mechanisms. Comprehensive systematic movement disorder evaluations were carried out on patients with this disorder. Methods: Patients born from 1995 to 2007 were identified from the Danish Cerebral Palsy Register and referrals to the neuropaediatric centre, Rigshospitalet, Copenhagen. They were classified by gross motor function, manual functional ability, communication ability, dystonia and spasticity. Electromyography was carried out on the upper and lower limbs. Magnetic resonance imaging scans were revised, and aetiological searches for underlying genetic disorders were performed. Results: We investigated 25 patients with dyskinetic CP at a mean age of 11.7 years. Dystonia, spasticity and rigidity were found in the upper limbs of 21, four and six children, respectively, and in the lower limbs of 18, 18 and three children. The mean total Burke-Fahn-Marsden score for dystonia was 45.02, and the mean Disability Impairment Scale level was 38% for dystonia and 13% for choreoathetosis. Sustained electromyography activity was observed in 20/25 children. Stretching increased electromyography activity more in children with spasticity. There were 10 re-classifications. Conclusion: The children had heterogenic characteristics, and 40% were reclassified after systematic movement disorder evaluation.

AIM: To investigate reasons for the declining prevalence of cerebral palsy (CP) in children born at term in Denmark by evaluating obstetric and neonatal factors associated with CP, and their changes over time.METHOD: In this cohort study, we included 987 495 children (504 600 [51.1%] males and 482 895 [48.9%] females) born after 37 completed gestational weeks during birth years 1997 to 2013. Risk ratios of CP for each factor were calculated with log-binominal regression analyses. Significant factors were evaluated concerning their development in prevalence over time.RESULTS: In the antenatal period, there were significant associations with an increased risk of CP and high maternal body mass index (BMI), smoking during pregnancy, nulliparity, male sex, gestational age, and low birthweight. In the study period, fewer females smoked during pregnancy and fewer children were born post-term, dropping from 22.6% to 11.4% and 9.4% to 2.5% respectively. Conversely, the proportion of females with high BMI increased. Most significant risk factors were found in the neonatal period, with an increase in children with diagnosed birth defects and children admitted to neonatal care.INTERPRETATION: Reasons for the declining prevalence of CP appear to be multifactorial and likely include the decline in maternal smoking and children born post-term along with centralization and advances in neonatal treatment.

AIM: To analyse trends in prevalence and severity of cerebral palsy (CP) in Denmark in birth years 1999-2007 and compare with previous periods.METHOD: Data has been collected uniformly in the Danish cerebral palsy national register nationwide since 1995. Rates in the time periods 1999-2001, 2002-2004 and 2005-2007 covering 585,393 births were analysed by gestational age and subtypes.RESULTS: Total number of CP cases in the period was 1165. The overall prevalence of CP decreased significantly from 2.1 in 1999-2001 to 1.8 in 2005-2007 per 1000 livebirths (p = 0.022). The decline was only significant for children born at term (p = 0.007) but not for the preterm (p = 0.44). The decline in children born at term was based on a decrease in bilateral spastic CP (n = 117 in years 1999-2001 and n = 59 in 2005-2007). Multidisciplinary obstetric skills training with neonatal resuscitation in Denmark was initiated in 2003 and timely associated with the decrease. The prevalence of unilateral spastic CP the prevalence did not change, but in the two last time periods more children had a right-sided than left-sided unilateral spastic CP.CONCLUSION: The decline in rate of CP seen in 2005-2007 as compared to 1999-2001 was mainly based on fewer cases of severe spastic CP in term infants. We hypothesize that improved neonatal resuscitation in the delivery room may be partly responsible for the decrease. In premature children the decline was not significant in this time period, but has been dramatically decreasing in the years before the time period here analysed.

We report detailed functional analyses and genotype-phenotype correlations in 433 individuals carrying disease-causing variants in SCN8A, encoding the voltage-gated Na+ channel NaV1.6. Five different clinical subgroups could be identified: 1) Benign familial infantile epilepsy (BFIE) (n=17, normal cognition, treatable seizures), 2) intermediate epilepsy (n=36, mild ID, partially pharmacoresponsive), 3) developmental and epileptic encephalopathy (DEE, n=191, severe ID, majority pharmacoresistant), 4) generalized epilepsy (n=21, mild to moderate ID, frequently with absence seizures), and 5) affected individuals without epilepsy (n=25, mild to moderate ID). Groups 1-3 presented with early-onset (median: four months) focal or multifocal seizures and epileptic discharges, whereas the onset of seizures in group 4 was later (median: 39 months) with generalized epileptic discharges. The epilepsy was not classifiable in 143 individuals. We performed functional studies expressing missense variants in ND7/23 neuroblastoma cells and primary neuronal cultures using recombinant tetrodotoxin insensitive human NaV1.6 channels and whole-cell patch clamping. Two variants causing DEE showed a strong gain-of-function (GOF, hyperpolarising shift of steady-state activation, strongly increased neuronal firing rate), and one variant causing BFIE or intermediate epilepsy showed a mild GOF (defective fast inactivation, less increased firing). In contrast, all three variants causing generalized epilepsy induced a loss-of-function (LOF, reduced current amplitudes, depolarising shift of steady-state activation, reduced neuronal firing). Including previous studies, functional effects were known for 165 individuals. All 133 individuals carrying GOF variants had either focal (76, groups 1-3), or unclassifiable epilepsy (37), whereas 32 with LOF variants had either generalized (14), no (11) or unclassifiable (5) epilepsy; only two had DEE. Computational modeling in the GOF group revealed a significant correlation between the severity of the electrophysiological and clinical phenotypes. GOF variant carriers responded significantly better to sodium channel blockers (SCBs) than to other anti-seizure medications, and the same applied for all individuals of groups 1-3.In conclusion, our data reveal clear genotype-phenotype correlations between age at seizure onset, type of epilepsy and gain- or loss-of-function effects of SCN8A variants. Generalized epilepsy with absence seizures is the main epilepsy phenotype of LOF variant carriers and the extent of the electrophysiological dysfunction of the GOF variants is a main determinant of the severity of the clinical phenotype in focal epilepsies. Our pharmacological data indicate that SCBs present a therapeutic treatment option in early onset SCN8A-related focal epilepsy.

Preterm infants have an increased risk of neurodevelopmental disorders. We established a direct quantitative comparison of the association between the degree of prematurity and three different neurodevelopmental disorders.In this cohort study, we combined data from 995,498 children in the Danish Medical Birth Register, from birth years 1997-2013, with information on cerebral palsy, epilepsy, and special educational needs. We estimated the gestational week-specific prevalence and risk for each of the disorders.The risk ratio of cerebral palsy at gestational weeks 21-24, compared to term birth, was more than ten times higher than for the two other disorders. The prevalence of epilepsy and special educational needs declined almost parallel, with 9.2% (4.6%-13.5%) and 12.5% (11.2%-13.7%), respectively, per week of gestation toward term birth. Cerebral palsy did not decline similarly: from gestational weeks 21-24 until week 29 the prevalence declined insignificantly by 0.6% (-11.1%-11.0%) per week; whereas from week 29 until term, the prevalence declined markedly by 36.7% (25.9%-45.9%) per week.The prevalence and risk of cerebral palsy are affected differently by the degree of prematurity compared with epilepsy and special educational needs, possibly reflecting important differences in cerebral pathophysiology.For each week of gestation toward term birth, there was a clear log-linear decline in the prevalence of early childhood epilepsy and special educational needs. In contrast, the risk of cerebral palsy was high at the earliest gestational age, and the prevalence did not decline significantly until gestational week 29, from where it declined notably by nearly 40% for each week of gestation until term birth. Our results indicate important differences in the pathophysiological processes that associate preterm birth with these three neurodevelopmental disorders.

Aim To quantify and analyse the prevalence and clinical features of cerebral palsy (CP) in Denmark for birth years 2008–2013 and compare results with previous periods. Method A nationwide register-based study covering all children with a confirmed diagnosis of CP born in Denmark. Information about CP subtype, aetiology and severity was collected from the Cerebral Palsy Follow-up Program and supplemented from medical files. Data from the Danish Medical Birth Register was included, and the results were compared to previous data from the Danish National Cerebral Palsy Register. Prevalence per 1000 live births and proportions were analysed using the Cochran-Armitage test for trend. Results The period covered 368,618 live births and 636 children with CP, making the overall prevalence for the period 1.73 per 1000 live births. This was significantly lower than the prevalence of 1.99 for the previous period 1999–2007 (p = 0.004). The decline in prevalence between the two periods was mainly due to a decrease in children with bilateral spastic and dyskinetic CP born after 37 gestational weeks. The decline in prevalence was accompanied by a smaller proportion of children with associated impairment. Conclusion We found a decrease in prevalence and severity in CP among Danish children. The decline was most pronounced in children born after 37 gestational weeks with severe subtypes of CP. National guidelines that recommend induction of labour before the completion of week 42 and therapeutic hypothermia for term neonates with hypoxic-ischaemic encephalopathy, may have contributed to the decline.

Ion channel mutations can cause distinct neuropsychiatric diseases. We first studied the biophysical and neurophysiological consequences of four mutations in the human Na+ channel gene SCN8A causing either mild (E1483K) or severe epilepsy (R1872W), or intellectual disability and autism without epilepsy (R1620L, A1622D). Only combined electrophysiological recordings of transfected wild-type or mutant channels in both neuroblastoma cells and primary cultured neurons revealed clear genotype-phenotype correlations. The E1483K mutation causing mild epilepsy showed no significant biophysical changes, whereas the R1872W mutation causing severe epilepsy induced clear gain-of-function biophysical changes in neuroblastoma cells. However, both mutations increased neuronal firing in primary neuronal cultures. In contrast, the R1620L mutation associated with intellectual disability and autism-but not epilepsy-reduced Na+ current density in neuroblastoma cells and expectedly decreased neuronal firing. Interestingly, for the fourth mutation, A1622D, causing severe intellectual disability and autism without epilepsy, we observed a dramatic slowing of fast inactivation in neuroblastoma cells, which induced a depolarization block in neurons with a reduction of neuronal firing. This latter finding was corroborated by computational modelling. In a second series of experiments, we recorded three more mutations (G1475R, M1760I, G964R, causing intermediate or severe epilepsy, or intellectual disability without epilepsy, respectively) that revealed similar results confirming clear genotype-phenotype relationships. We found intermediate or severe gain-of-function biophysical changes and increases in neuronal firing for the two epilepsy-causing mutations and decreased firing for the loss-of-function mutation causing intellectual disability. We conclude that studies in neurons are crucial to understand disease mechanisms, which here indicate that increased or decreased neuronal firing is responsible for distinct clinical phenotypes.

AIM: We aimed at describing clinical findings in children with dyskinetic as compared to bilateral spastic cerebral palsy (CP).METHODS: Data was extracted from the Danish nationwide CP register. Participants were born in 1999-2007 and were 5-6 years at ascertainment.RESULTS: The total number of CP cases was 1,165 of which 92 had dyskinetic and 540 bilateral spastic CP. Prevalence of dyskinetic CP was 0.16 per 1000 live-births. In participants with dyskinetic compared to bilateral spastic CP, there was more frequently an Apgar level less than five at five minutes (22.7% versus 11.2%) and neonatal seizures (43.5% versus 28.5%), but less respiratory deficiency, hyperbilirubinaemia, and sepsis. Impairment based on gross motor function classification was more severe in dyskinetic CP (level III-V 90.0% versus 66.0%). In dyskinetic CP there was a high rate of reduced developmental quotient (68.1%), visual impairment (39.3%), and epilepsy (51.6%). Basal ganglia lesions were more prevalent in dyskinetic compared to bilateral spastic CP (27.7% vs. 12.8%).CONCLUSION: Cases of dyskinetic CP had overlapping clinical features with cases of bilateral spastic CP, but differed significantly in several perinatal risk factors. The children with dyskinetic CP had experienced more peri- or neonatal adverse events, and neurodevelopmental impairment was severe. This article is protected by copyright. All rights reserved.

Infantile spasms is one of the epileptic encephalopathies. These have a variety of established etiological factors, but in a subset of cases, the underlying cause is not evident despite extensive investigations. These cases are referred to as idiopathic or cryptogenic infantile spasms. We report on two cases where the etiology was established by high-resolution array-comparative genomic hybridization as a de novo deletion in the STXBP1 gene. We propose to include high-resolution array-comparative genomic hybridization as a part of the diagnostic evaluation program in patients with infantile spasms with an unknown etiology. Establishment of the etiology has important implications for prognosis and genetic counseling.

AimGerm cell tumours (GCTs) are a rare heterogeneous tumour group derived from primordial germ cells, which can be benign or malignant and occur in the gonads or extragonadally. This study mapped the paediatric GCTs in Denmark from 1984 to 2013 to study the incidence and outcome.MethodsWe identified paediatric GCTs from the Danish Childhood Cancer and National Pathology Registries and reviewed the case records for patient characteristics, tumour characteristics and clinical outcome.ResultsWe identified 403 (71% female) paediatric GCTs and the crude incidence was 1.43 per 100 000. Of these, 79 (20%) were malignant, 39 (10%) were potentially malignant and 285 (70%) were benign. Extragonadal GCTs (39%) were mainly observed in early childhood and were predominately sacrococcygeal teratomas. Gonadal GCTs (61%) in late childhood were most frequently mature teratomas in the ovaries. Nearly all patients underwent surgery. Of the malignant tumours, 62% were treated with chemotherapy. Radiotherapy was only administered to intracranial GCTs. In the cohort, 12 patients died (3%).ConclusionPaediatric GCTs in Denmark were mainly benign and mortality was low, even for malignant tumours. We identified a peak of extragonadal GCTs in early childhood and a peak of gonadal GCTs in late childhood, which was comparable to previous reports.

Paediatric germ cell tumours (GCTs) are rare and account for less than 3 % of childhood cancers. Like adult GCTs, they probably originate from primordial germ cells, but the pattern of histopathological types is different, and they occur predominantly in extragonadal sites along the body midline. Because they are rare, histology of paediatric GCTs is poorly documented, and it remains unclear to what extent they differ from adult GCTs. We have analysed 35 paediatric germ cell tumours and 5 gonadal sex-cord stromal tumours from prepubertal patients aged 0-15 years, to gain further knowledge, elaborate on clinical-pathological associations and better understand their developmental divergence. The tumours were screened for expression of stemness-related factors (OCT4, AP-2γ, SOX2), classical yolk sac tumours (YSTs; AFP, SALL4), GCTs (HCG, PLAP, PDPN/D2-40), as well as markers for sex-cord stromal tumour (PDPN, GATA4). All YSTs expressed AFP and SALL4, with GATA4 present in 13/14. The majority of teratomas expressed SOX2 and PDPN, whereas SALL4 was found in 8/13 immature teratomas. Adult seminoma markers AP-2γ, OCT4, SALL4 and PDPN were all expressed in dysgerminoma. We further report a previously unrecognised pathogenetic relationship between AFP and SALL4 in YST in that different populations of YST cells express either SALL4 or AFP, which suggests variable differentiation status. We also show that AP-2γ is expressed in the granulosa layer of ovarian follicles and weakly expressed in immature but not in mature granulosa cell tumours. Our findings indicate that the expression pattern of these antigens is similar between paediatric and adult GCTs, even though they develop along different developmental trajectories.

The DNA damage response (DDR) machinery becomes commonly activated in response to oncogenes and during early stages of development of solid malignancies, with an exception of testicular germ cell tumors (TGCTs). The active DDR signaling evokes cell death or senescence but this anti-tumor barrier can be breached by defects in DDR factors, such as the ATM-Chk2-p53 pathway, thereby allowing tumor progression. The DDR barrier is strongly activated in brain tumors, particularly gliomas, due to oxidative damage and replication stress. Here, we took advantage of rare human primary intracranial germ cell tumors (PIGCTs), to address the roles of cell-intrinsic factors including cell of origin, versus local tissue environment, in the constitutive DDR activation in vivo. Immunohistochemical analysis of 7 biomarkers on a series of 21 PIGCTs (germinomas and other subtypes), 20 normal brain specimens and 20 glioblastomas, revealed the following: i) The overall DDR signaling (γH2AX) and activation of the ATM-Chk2-p53 pathway were very low among the PIGCTs, reminiscent of TGCTs, and contrasting sharply with strong DDR activation in glioblastomas; ii) Except for one case of embryonal carcinoma, there were no clear aberrations in the ATM-Chk2-p53 pathway components among the PIGCT cohort; iii) Subsets of PIGCTs showed unusual cytosolic localization of Chk2 and/or ATM. Collectively, these results show that PIGCTs mimic the DDR activation patterns of their gonadal germ cell tumor counterparts, rather than the brain tumors with which they share the tissue environment. Hence cell-intrinsic factors and cell of origin dictate the extent of DDR barrier activation and also the ensuing pressure to select for DDR defects. Our data provide conceptually important insights into the role of DNA damage checkpoints in intracranial tumorigenesis, with implications for the differential biological responses of diverse tumor types to endogenous stress as well as to genotoxic treatments such as ionizing radiation or chemotherapy.

Pediatric germ cell tumors (GCTs) are rare neoplasms arising predominantly in the gonads and sacrococcygeal, mediastinal, and intracranial localizations. In this article, we review current knowledge of pathogenesis of pediatric GCTs, which differs from adult/adolescent GCTs. One distinctive feature is the absence of a progenitor stage, such as carcinoma in situ or gonadoblastoma, which are seen in adult/adolescent GCTs, except spermatocytic seminoma. The primordial germ cell (PGC) is the suggested origin of all GCTs, with variations in histology reflecting differentiation stage. Expression of pluripotency transcription factors OCT-3/4, NANOG, and AP-2γ in germinomas/seminomas/dysgerminomas is consistent with retaining a germ cell phenotype. Teratomas, in contrast, develop through a pathway of aberrant somatic differentiation of immature germ cells, and the yolk sac tumors and choriocarcinomas result from abnormal extraembryonic differentiation. In pediatric GCTs, origin is suggested at an earlier developmental stage because of predisposing genetic factors, although responsible genes remain largely unknown. Some extragonadal GCTs have been linked to overexpression of the KIT/KITLG system, allowing for survival of aberrantly migrated ectopic PGCs. Infant gonadal/sacrococcygeal GCTs may be caused by apoptosis-related pathways, consistent with an association with polymorphisms in BAK1. Although recent advances have identified candidate pathways, further effort is needed to answer central questions of pathogenesis of these fascinating tumors.

This review focuses on the molecular characteristics and development of rare malignant ovarian germ cell tumors (mOGCTs). We provide an overview of the genomic aberrations assessed by ploidy, cytogenetic banding, and comparative genomic hybridization. We summarize and discuss the transcriptome profiles of mRNA and microRNA (miRNA), and biomarkers (DNA methylation, gene mutation, individual protein expression) for each mOGCT histological subtype. Parallels between the origin of mOGCT and their male counterpart testicular GCT (TGCT) are discussed from the perspective of germ cell development, endocrinological influences, and pathogenesis, as is the GCT origin in patients with disorders of sex development. Integrated molecular profiles of the 3 main histological subtypes, dysgerminoma (DG), yolk sac tumor (YST), and immature teratoma (IT), are presented. DGs show genomic aberrations comparable to TGCT. In contrast, the genome profiles of YST and IT are different both from each other and from DG/TGCT. Differences between DG and YST are underlined by their miRNA/mRNA expression patterns, suggesting preferential involvement of the WNT/β-catenin and TGF-β/bone morphogenetic protein signaling pathways among YSTs. Characteristic protein expression patterns are observed in DG, YST and IT. We propose that mOGCT develop through different developmental pathways, including one that is likely shared with TGCT and involves insufficient sexual differentiation of the germ cell niche. The molecular features of the mOGCTs underline their similarity to pluripotent precursor cells (primordial germ cells, PGCs) and other stem cells. This similarity combined with the process of ovary development, explain why mOGCTs present so early in life, and with greater histological complexity, than most somatic solid tumors.

Purpose Epilepsy surgery is performed based on the assumption that medical refractory epilepsy will continue. Rarely seizure freedom occurs before surgery is performed, while the patient is being evaluated as an epilepsy surgery candidate. The aim of this study was to describe the number of children withdrawn from an epilepsy surgery programme due to unexpected seizure improvement. Methods We retrospectively studied 173 children under 18 years with medical refractory epilepsy referred for epilepsy surgery between 1996 and 2010. Medical records were reviewed in 2012 and 2015. Results At the first evaluation point in 2012, 13 patients were withdrawn from the epilepsy surgery programme due to unexpected marked improvement. In 2015, 6 of them were still seizure free. They had unexpected seizure freedom due to change in AED treatment (n=3) or after a febrile episode (n=3). The mean number of years they had had seizures was 3.4 years (range 0.6–6.2 years) and the number of seizures at inclusion was 209 per month (range 6–750 per month). The duration of follow-up was 6.6 years after inclusion into the epilepsy surgery programme (range 4.0–13.0 years). The aetiology of the epilepsy for these patients was heterotopia (n=1), focal cortical dysplasia (n=3), infarction (n=1) and unknown, with normal MRI (n=1). They all had an IQ in the normal range. Two of the remaining 7 children were operated later. Conclusion Unexpected seizure control may occur during epilepsy surgery evaluation.

Testicular germ-cell tumors occur primarily in young individuals, and the tumors in this age group (seminomas or nonseminomas) are derived from a preinvasive precursor cell called carcinoma in situ (CIS) or intratubular germ-cell neoplasia. These tumors have been a growing problem, especially in highly developed industrialized countries. A hypothesis was put forward that CIS originates from arrested fetal germ cells, thus testicular cancer is a developmental disease of germ-cell differentiation. This notion was supported by comparative studies of the gene expression at the protein and RNA level, which demonstrated a close similarity of CIS to primordial germ cells and gonocytes with many features of embryonic stem cells. The arrest of germ-cell differentiation is thus the key first event, which may be followed by malignant transformation and overt germ-cell cancer in young adult age, usually after puberty. In most cases the arrest/delay of germ-cell differentiation is caused by testicular dysgenesis, a multifactorial and complex syndrome that has a broad spectrum of phenotypes ranging from moderate impairment of spermatogenesis to severe disorders of sexual development and differentiation. The most severe cases are a result of inherited genetic aberrations, but the etiology of the common sporadic testicular cancer must involve environmental factors, including maternal lifestyle and possibly an early exposure to endocrine disruptors. The effects of environmental factors are likely modulated by genomic variation (polymorphisms), thus explaining the individual susceptibility and population-level differences in the incidence of testicular cancer.

The testicular dysgenesis syndrome (TDS) hypothesis proposes that a proportion of the male reproductive disorders-cryptorchidism, hypospadias, infertility and testicular cancer-may be symptoms of one underlying developmental disease, TDS, which is most likely a result of disturbed gonadal development in the embryo. TDS may be caused by genetic factors, environmental/life-style factors, or a combination of both. Some rare disorders of sex development of genetic origin are among the best-known examples of severe TDS. Among the environmental and life-style factors that are suspected to influence the hormonal milieu of the developing gonad are the endocrine disrupters. A prenatal exposure to commonly used chemicals, e.g. phthalates, may result in a TDS-like phenotype in rats. Currently, this animal model is the best model for TDS. In humans the situation is much more complex, and TDS exists in a wide range of phenotypes: from the mildest and most common form, in which impaired spermatogenesis is the only symptom, to the most severe cases, in which the patient may develop testicular cancer. It is of great importance that clinicians in different specialties treating patients with TDS are aware of the association between the different symptoms.

Testicular germ cell tumours have a favourable prognosis if detected early, but are potentially lethal in a subset of patients. Multi-modality treatment is often necessary, thus the preferable time of diagnosis is at the pre-invasive, but unfortunately often asymptomatic precursor stage of carcinoma in situ (CIS). This review describes current possible approaches for the detection of CIS. At present, an open testicular biopsy is the only definitive way of establishing the presence of CIS. The tissue section should be of an adequate size, be properly fixed, and evaluation be supported by at least one solid immunohistochemical marker, for example PLAP, OCT-3/4 or AP-2gamma. Determination of who should be offered testicular biopsies is based on clinical and ultrasonic examination along with the evaluation of risk factors. A surgical biopsy is an invasive procedure with potential complications, although rare. Therefore, a noninvasive and equally reliable method is needed. Testicular ultrasound is risk-free, painless and at present the only noninvasive method of aid for andrologists when CIS is suspected. The presence of testicular microlithiasis is, in some cases, indicative of pre-malignant changes, especially in males with additional risk factors. Promising results have recently been obtained with a novel noninvasive detection method based on immunocytological AP-2gamma-staining of CIS cells in semen. This method could be a supporting method in andrology centres where careful follow-up is possible. In conclusion, one difficulty is to determine in which males CIS should be suspected; secondly, there does not as yet exist an optimal noninvasive method of diagnosis that is more acceptable than an open surgical biopsy.

Carcinoma in situ (CIS) testis is the precursor stage for the majority of testicular germ cell tumours (TGCT). Infertility is one of the conditions known to predispose to TGCT, but based on scarce existing data, the prevalence of CIS in this risk group was estimated at only approximately 1%. To establish more objective data, we investigated retrospectively the prevalence of CIS based on testicular biopsies performed in a well-defined group of subfertile males. We included 453 patients who had testicular biopsies performed for infertility reasons during 1995-2005 at the Copenhagen University Hospital (Rigshospitalet). Biopsies were evaluated by two experienced observers independently. CIS was detected in 10 individuals, of whom three had bilateral CIS, corresponding to a prevalence of 2.2% (95% CI 1.1-4.0%). This is greater than the estimated risk of 0.45% for the age- and birth cohort-matched general Danish population. All patients with CIS testis had severe oligozoospermia (/=2.06 million/mL). We confirmed that a thorough examination of men suffering from subfertility/infertility can identify those with an increased risk for testicular neoplasia and recommend performing bilateral biopsies, especially in the subpopulation of men with atrophic testicles, severe oligozoospermia and/or irregular ultrasonic pattern of their testicles.

Summary Germ cell tumours (GCTs) are a complex entity. Current areas of attention include early detection and avoidance of unnecessary over-treatment. Novel findings regarding diagnosis of GCTs located in various anatomical sites are described, particularly testicular GCTs and their common progenitor, carcinoma in situ (CIS). Recognition of CIS enables intervention before tumour development, but nevertheless, testicular GCTs are sporadically diagnosed at the pre-invasive stage where minimal treatment is necessary. As presence of CIS is asymptomatic, a simple screening method is needed when CIS is suspected (i.e. in males investigated for infertility). To develop approaches for early detection CIS gene expression studies have been performed showing many similarities with embryonic stem cells with confirmation of established markers (i.e. PLAP, OCT-3/4, KIT) and identification of novel markers (i.e. AP-2γ, NANOG). We have reported a very promising new approach of AP-2γ (or OCT3/4) based immunocytological semen analysis (specificity 93.6%, sensitivity 54.5%). Comparative studies of gonadal/extragonadal GCTs have revealed resemblance pointing towards similar, but not identical, origins. Moreover, infertility and testicular cancer are connected in the ‘Testicular Dysgenesis Syndrome’ and 25% of contralateral testes from testicular GCT patients harbour dysgenetic features, including impaired spermatogenesis. Thus, recent data have provided potential diagnostic tools including CIS detection in semen, microarray-based tumour classification, additional serological GCT markers, and novel stem cell markers for immunohistochemical diagnosis of gonadal and extragonadal GCTs. Many CIS candidate genes are yet uninvestigated, and information from these could increase knowledge about CIS tumour initiation/progression and be used for optimisation of a non-invasive detection method.

E-cadherin (CDH1) is a transmembrane glycoprotein involved in cellular adhesion. In our recent microarray studies of testicular germ cell tumours (TGCTs) and the common precursor carcinoma in situ (CIS), CDH1 mRNA was highly expressed in CIS and embryonal carcinoma. It has previously been reported that the CDH1 protein is not expressed in CIS. To resolve the discrepancy, we performed a detailed analysis of the expression of CDH1 mRNA and protein in a series of normal and neoplastic testes. High expression of CDH1 mRNA in CIS was confirmed by real-time PCR and in situ hybridisation. At the protein level, however, CDH1 was only detected with one of three tested antibodies, but Western blotting analysis with this antibody showed additional bands, suggesting unspecific staining. The levels of a CDH1 protein fragment in serum samples from 58 patients with TGCTs were analysed by ELISA; we found significantly higher levels in patients with advanced disease (stage II/III) when compared to healthy individuals and patients with stage I TGCT. In conclusion, despite high mRNA levels, the CDH1 protein is not expressed in CIS, suggesting translational suppression of CDH1 protein expression. CDH1 serum levels may be a serological marker for staging of TGCT patients.

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

The carcinoma in situ (CIS) cell is the common precursor of nearly all testicular germ cell tumours (TGCT). In a previous study, we examined the gene expression profile of CIS cells and found many features common to embryonic stem cells indicating that initiation of neoplastic transformation into CIS occurs early during foetal life. Progression into an overt tumour, however, typically first happens after puberty, where CIS cells transform into either a seminoma (SEM) or a nonseminoma (N-SEM). Here, we have compared the genome-wide gene expression of CIS cells to that of testicular SEM and a sample containing a mixture of N-SEM components, and analyse the data together with the previously published data on CIS. Genes showing expression in the SEM or N-SEM were selected, in order to identify gene expression markers associated with the progression of CIS cells. The identified markers were verified by reverse transcriptase-polymerase chain reaction and in situ hybridisation in a range of different TGCT samples. Verification showed some interpatient variation, but combined analysis of a range of the identified markers may discriminate TGCT samples as SEMs or N-SEMs. Of particular interest, we found that both DNMT3B (DNA (cytosine-5-)-methyltransferase 3 beta) and DNMT3L (DNA (cytosine-5-)-methyltransferase 3 like) were overexpressed in the N-SEMs, indicating the epigenetic differences between N-SEMs and classical SEM.

Purpose: We compared clinical and histological data regarding the contralateral testicle in a population of men diagnosed with testicular germ cell cancer to find features associated with an increased risk of bilateral neoplasia.Materials and Methods: Clinical data obtained from the records of 79 men seen during the period 1998 to 2001 included history, physical and ultrasound examination, and the results of semen and hormonal analysis. Contralateral testicular biopsies were obtained from all men and analyzed for carcinoma in situ (CIS), microcalcifications (microliths) and other signs of testicular dysgenesis.Results: Ultrasound examination of the contralateral testicle was performed in 64 of the cases. The echo pattern was judged normal in 30 cases, slightly irregular in 22, irregular in 3 and showed microlithiasis in 9. The frequency of microlithiasis seen on ultrasound was significantly higher among patients with CIS (p

Durant les dernieres decennies, le cancer du testicule a cellules germinales a augmente dans de nombreux pays a travers le monde. Ces dernieres annees, l’attention a ete attiree par les nombreuses recherches revelant des tendances negatives dans d’autres aspects de la sante reproductive masculine, comme une frequence elevee et probablement en augmentation du testicule non descendu et de l’hypospadias, une qualite du sperme declinante, et une demande apparemment croissante de reproduction assistee pour infecondite masculine. Le present article resume les preuves qui sont en faveur d’un nouveau concept, a savoir que ces anomalies de la reproduction masculine pourraient constituer les signes d’une entite sous-jacente, le Syndrome de Dysgenesie Testiculaire (SDT). Ce syndrome, du a des retards non specifiques et a des aberrations du developpement testiculaire precoce, pourrait devenir de plus en plus commun en raison de facteurs deleteres de l’environnement et du mode de vie qui perturbent le developpement gonadique. Les variations geographiques et ethniques de l’incidence des differentes formes de SDT peuvent s’expliquer soit par des differences d’exposition aux facteurs adverses, soit par des variations de la susceptibilite genetique a ces facteurs.

Alternating hemiplegia of childhood (AHC) is a rare neurodevelopmental disorder characterized by early-onset recurrent distinctive hemiplegic episodes commonly accompanied by other paroxysmal features and developmental impairment. De novo mutations in ATP1A3 were recently identified as a genetic cause of AHC. To describe the entire Danish cohort of paediatric AHC patients we approached neuropaediatricians nationwide. All currently acknowledged Danish patients ≤16 years with AHC were genetically tested and seen by the same child neurologist (PU). Ten patients; seven girls and three boys were identified. Mean present age was 10.0 years (range 1-16). Mean age at presentation was 7.4 months (range 1-18 months). Sequencing of ATP1A3 in all ten patients revealed a pathogenic mutation in seven. Two females with moderate psychomotor impairment were heterozygous for the known p.G947R mutation, whereas one severely retarded boy was heterozygous for the common p.E815K mutation. The prevalent p.D801N mutation was identified in two moderate to severely retarded children. Interestingly, in a set of monochorionic male twins a novel p.D801E mutation was identified, underscoring that the asparagine at position 801 is a mutation hotspot. Three girls aged 5-13 years did not reveal any ATP1A3 mutations. They were rather mildly clinically affected and displayed a normal or near-normal psychomotor development. This is the first study of AHC in the Danish paediatric population. The patients harboured a wide range of psychomotor difficulties. Patients with no mutation detected tended to be less severely affected. Prevalence was approximately 1 per 100,000 children.

Does more than one biopsy of the contralateral testis in men with a germ cell tumor add value?

The pathogenesis and mechanisms behind the degeneration of the seminiferous tubules in testes of subjects with Klinefelter syndrome (KS) are yet unknown.The objective of this prospective clinical study was to characterize the testicular degeneration process during puberty in boys with KS by describing the immunoexpression of some developmentally regulated markers of testis maturation in relation to serum levels of reproductive hormones.This study was conducted at a university central hospital pediatric referral endocrinology outpatient clinic.Patients consisted of 14 boys with KS aged 10.1 to 14.0 yr.Main outcome measures were immunoexpression of germ cell differentiation markers (AP-2gamma, CHK2, OCT-3/4, NY-ESO-1, MAGE-A4) and androgen action-related proteins [androgen receptor (AR), anti-Müllerian hormone (AMH), MIC2, inhibin B; alpha- and betaB-subunits] in testicular biopsies of boys with KS in relation to serum reproductive hormone levels.In boys with KS, gonocytes differentiated to the spermatogonium stage, but no spermatocytes were visible. Despite this, down-regulation of AMH expression in the Sertoli cells occurred concomitantly with decreasing serum AMH levels. Expression of inhibin alpha- and betaB-subunits appeared in the biopsies even when circulating inhibin B levels were undetectable. In the boys with KS compared with age-matched controls, the proportion of Sertoli cell nuclei expressing AR was smaller and cytoplasmic staining of Sertoli cells was constantly present.We showed with several testis-specific markers in KS that gonocytes differentiate to spermatogonia and that the degeneration of the testes accelerates at the onset of puberty. Altered immunoexpression of AR indicates that a relative androgen deficiency, at least at the testicular level, develops in boys with KS during puberty.

Background Testicular germ cell tumours (TGCTs) originate from a common precursor, carcinoma in situ (CIS). Diagnosis at the CIS stage is desirable as it minimizes the necessary treatment. A detailed clinical evaluation of an approach to detect CIS cells in the ejaculate using primordial germ cell/gonocyte markers is presented. Methods Immunocytological staining for AP-2gamma [and in some cases, OCT-3/4, NANOG or placental alkaline phosphatase (PLAP)] was performed in semen samples from 294 infertile patients and 209 patients with TGCTs or other diseases. Results Presence of AP-2gamma-stained cells was detected in 50% of participants with CIS and in 33.9% of TGCT patients before treatment (non-seminomas: 56.6%, seminomas: 17.4%). OCT-3/4 results were similar to those of AP-2gamma, whereas NANOG and PLAP stainings were unsuitable. Sensitivity was 54.5% for participants harbouring pre-invasive CIS but reduced in participants with overt TGCTs, perhaps because of obstruction. Assay specificity was 93.6%, positive predictive value (PPV) 83.3% and negative predictive value (NPV) 60.3%. Conclusions Immunocytological semen analysis based on expression of fetal germ cell markers in exfoliated cells has auxiliary diagnostic value, as it detects some patients with CIS/incipient tumour, but a negative result does not exclude TGCT. Further effort is needed to improve this assay, for example, by employing a more sensitive biochemical method of detection.

Testicular biopsy is a crucial assessment in reproductive practice with diagnostic and prognostic importance for assisted reproductive technologies (ARTs) and risk of testicular neoplasia. Endocrine and genetic tests cannot reliably distinguish obstructive azoospermia (OA) from non-obstructive azoospermia (NOA) or predict recovery of mature spermatids by testicular sperm extraction (TESE). Currently, divergent histological reporting systems and the use of imprecise terminology seriously degrade the value of the literature on TESE recovery rates and hamper evaluation of treatments and research on genotype-phenotype relationships. The rising incidence of testis cancer and carcinoma in situ (CIS), especially in infertile populations, requires that every effort be made for its early detection. We provide a systematic approach to the histological classification of spermatogenic disorders and detection of CIS in adult patients. We evaluate a large consecutive series of bilateral biopsies from infertile men and report (i) the frequency of bilateral or discordant patterns that supports the use of bilateral biopsy for comprehensive evaluation and (ii) a high prevalence of mixed patterns, particularly within the hypospermatogenesis classification, that helps account for reported success of TESE. We propose a new diagnosis code for testicular biopsies that addresses the needs of ART clinicians and allows data storage and retrieval of value in clinical practice and research.

Primary intracranial germ cell tumours are rare neoplasms that occur in children and adolescents. This study examined both the biology and the origin of these tumours, as it has been hypothesized that they originate from a totipotent primordial germ cell. We applied recent knowledge from gonadal germ cell tumours and analysed expression of a wide panel of stem cell-related proteins (C-KIT, OCT-3/4 (POU5F1), AP-2γ (TFAP2C), and NANOG) and developmentally regulated germ cell-specific proteins (including MAGE-A4, NY-ESO-1, and TSPY). Expression at the protein level was analysed in 21 children and young adults with intracranial germinomas and non-germinomas, contributing to a careful description of these unusual tumours and adding to the understanding of pathogenesis. Stem cell related proteins were highly expressed in intracranial germ cell tumours, and many similarities were detected with their gonadal equivalents, including a close similarity with primordial germ cells. A notable difference was the sex-specific expression of TSPY, a gene previously implicated in the origin of gonadoblastoma. TSPY was only detected in germ cell tumours in the central nervous system (CNS) from males, suggesting that it is not required for the initiation of malignant germ cell transformation. The expression of genes associated with embryonic stem cell pluripotency in CNS germ cell tumours strongly suggests that these tumours are derived from cells that retain, at least partially, an embryonic stem cell-like phenotype, which is a hallmark of primordial germ cells. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Testicular germ-cell tumours of young adults are derived from a pre-invasive intratubular lesion, carcinoma in situ (CIS). In a recent genome-wide gene expression screening using cDNA microarrays, we found PDPN over-expressed in CIS compared to normal adult testis. PDPN encodes podoplanin (Aggrus, human gp36, T1A-2), a transmembrane glycoprotein expressed in lymphatic endothelium and various solid tumours. To examine a potential role for PDPN in testicular neoplasms and during testicular development, we investigated its expression pattern during the development of human testis and in a series of testicular CIS, gonadoblastoma and overt germ-cell tumours. We established by RT-PCR and by immunohistochemistry with a gp36 antibody that PDPN mRNA and the protein product were expressed in testes with germ-cell neoplasms but not in the normal adult testis. We also found gp36 expression in early foetal gonocytes and immature Sertoli cells, similar to the expression pattern of M2A antigen, a previously identified marker for CIS and seminoma. This reinforced our previous proposal that M2A (D2-40) antigen was identical to gp36 (podoplanin, Aggrus, T1A-2). Our findings also suggest that podoplanin has a function in developing testis, most likely at the level of cell–cell interactions among pre-meiotic germ cells and immature Sertoli cells.

A rare case is presented where a dysgenetic testis with microinvasive carcinoma in situ (CIS, also known as intratubular germ cell neoplasm of unclassified type [IGCNU] and testicular intraepithelial neoplasia [TIN]) with microinvasion to rete testis and the interstitial tissue was found in a 32-year-old man presenting with mild scrotal pain and ultrasonic testicular microlithiasis. Knowledge of the association of ultrasound and CIS is important to diagnose patients at the stage prior to development of an overt germ cell tumor. The patient had three of four disorders considered symptoms of the testicular dysgenesis syndrome (TDS): a dysgenetic left testicle with CIS, a mild left-sided cryptorchidism (high positioned scrotal hypotrophic testis) and a slightly reduced semen quality. Therefore, it should be kept in mind that a patient with one TDS symptom may harbour the other, even CIS or testicular cancer. Accordingly, patients with one TDS symptom ought to be examined for the presence of the others, and if more that one is present, extra concern is warranted.

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