Residual disease after neoadjuvant chemoradiotherapy for oesophageal cancer: locations undetected by endoscopic biopsies in the preSANO trial.

BACKGROUND: Active surveillance has been proposed for patients with oesophageal cancer in whom there is a complete clinical response after neoadjuvant chemoradiotherapy (nCRT). However, endoscopic biopsies have limited negative predictive value in detecting residual disease. This study determined the location of residual tumour following surgery to improve surveillance and endoscopic strategies. METHODS: The present study was based on patients who participated in the prospective preSANO trial with adenocarcinoma or squamous cell carcinoma of the oesophagus or oesophagogastric junction treated in four Dutch hospitals between 2013 and 2016. Resection specimens and endoscopic biopsies taken during clinical response evaluations after nCRT were reviewed by two expert gastrointestinal pathologists. The exact location of residual disease in the oesophageal wall was determined in resection specimens. Endoscopic biopsies were assessed for the presence of structures representing the submucosal layer of the oesophageal wall. RESULTS: In total, 119 eligible patients underwent clinical response evaluations after nCRT followed by standard surgery. Residual tumour was present in endoscopic biopsies from 70 patients, confirmed on histological analysis of the resected organ. Residual tumour was present in the resection specimen from 27 of the other 49 patients, despite endoscopic biopsies being negative. Of these 27 patients, residual tumour was located in the mucosa in 18, and in the submucosa beneath tumour-free mucosa in eight. One patient had tumour in muscle beneath tumour-free mucosa and submucosa. CONCLUSION: Most residual disease after nCRT missed by endoscopic biopsies was located in the mucosa. Active surveillance could be improved by more sampling and considering submucosal biopsies.

ANTECEDENTES: Se ha propuesto un seguimiento activo para los pacientes con cáncer de esófago en los que se logra una respuesta clínica completa tras quimiorradioterapia neoadyuvante (neoadjuvant chemoradiotherapy, nCRT). Sin embargo, las biopsias endoscópicas tienen un valor predictivo limitado para detectar la enfermedad residual. En este estudio se evaluó la localización del tumor residual tras la cirugía para poder determinar estrategias de seguimiento y endoscópicas. MÉTODOS: Este estudio se basa en pacientes que participaron en el ensayo prospectivo preSANO (adenocarcinoma o carcinoma escamoso del esófago o unión esofagogástrica en cuatro hospitales de los Países Bajos entre 2013 y 2016). Los especímenes quirúrgicos, así como las biopsias endoscópicas efectuadas durante las evaluaciones de la respuesta clínica después de nCRT fueron revisadas por dos patólogos gastrointestinales expertos. En los especímenes de resección, se determinó la localización exacta de la enfermedad residual en la pared del esófago. Se evaluaron las biopsias endoscópicas para identificar estructuras que constituyeran la capa submucosa de la pared del esófago. RESULTADOS: En total, 119 pacientes elegibles fueron sometidos a evaluaciones de la respuesta clínica tras nCRT seguida de cirugía estándar. Se detectó tumor residual en las biopsias endoscópicas de 70 pacientes, luego confirmadas en la histología de la pieza extirpada. Se identificó tumor residual en la pieza de resección de 27 de los otros 49 pacientes, a pesar de que las biopsias endoscópicas fueron negativas. En estos 27 pacientes, 18 presentaban tumor residual en la mucosa y ocho pacientes en la submucosa mas allá de una mucosa libre de tumor. Un paciente tenía tumor en el músculo más allá de una mucosa y submucosa libres de tumor. CONCLUSIÓN: La mayoría de los casos de enfermedad residual tras nCRT que no se detectaron en las biopsias endoscópicas, se localizaban en la mucosa. El seguimiento activo podría mejorar con la toma de más muestras y considerando las biopsias submucosas.

The oncogene Gankyrin is expressed in testicular cancer and contributes to cisplatin sensitivity in embryonal carcinoma cells.

BACKGROUND: Testicular germ cell cancer (TGCC) develops from pre-malignant germ neoplasia in situ (GCNIS) cells. GCNIS originates from fetal gonocytes (POU5F1+/MAGE-A4-), which fail to differentiate to pre-spermatogonia (POU5F1-/MAGE-A4+) and undergo malignant transformation. Gankyrin is an oncogene which has been shown to prevent POU5F1 degradation and specifically interact with MAGE-A4 in hepatocellular carcinoma (HCC) cells. We aimed to investigate the role of Gankyrin in progression from gonocyte to pre-invasive GCNIS and subsequent invasive TGCC. METHODS: We determined Gankyrin expression in human fetal testicular tissue (gestational weeks 9-20; n = 38), human adult testicular tissue with active spermatogenesis (n = 9), human testicular tissue with germ cell maturation delay (n = 4), testicular tissue from patients with pre-invasive GCNIS (n = 6), and invasive TGCC including seminoma (n = 6) and teratoma (n = 7). Functional analysis was performed in-vitro by siRNA knock-down of Gankyrin in the NTera2 cells (derived from embryonal carcinoma). RESULTS: Germ cell expression of Gankyrin was restricted to a sub-population of prespermatogonia in human fetal testes. Nuclear Gankyrin was also expressed in GCNIS cells of childhood and adult pre-invasive TGCC patients, and in GCNIS from seminoma and non-seminoma patients. Cytoplasmic expression was observed in seminoma tumour cells and NTera2 cells. Gankyrin knock-down in NTera2 cells resulted in an increase in apoptosis mediated via the TP53 pathway, whilst POU5F1 expression was unaffected. Furthermore, Gankyrin knock-down in NTera2 cells increased cisplatin sensitivity with an increase in cell death (13%, p < 0.05) following Gankyrin knock-down, when compared to cisplatin treatment alone, likely via BAX and FAS. Our results demonstrate that Gankyrin expression changes in germ cells during normal transition from gonocyte to prespermatogonia. In addition, changes in Gankyrin localisation are associated with progression of pre-invasive GCNIS to invasive TGCC. Furthermore, we found that Gankyrin is involved in the regulation of NTera2 cell survival and that a reduction in Gankyrin expression can modulate cisplatin sensitivity. CONCLUSIONS: These results suggest that manipulation of Gankyrin expression may reduce the cisplatin dose required for the treatment of TGCC, with benefits in reducing dose-dependent side effects of chemotherapy. Further studies are required in order to assess the effects of modulating Gankyrin on GCNIS/TGCC using in vivo models.

Cellular origin of microRNA-371a-3p in healthy males based on systematic urogenital tract tissue evaluation.

BACKGROUND: The microRNA-371a-3p (miR-371a-3p) has been reported to be an informative liquid biopsy (serum and plasma) molecular biomarker for both diagnosis and follow-up of patients with a malignant (testicular) germ cell tumor ((T)GCT). It is expressed in all histological cancer elements, with the exception of mature teratoma. However, normal testis, semen, and serum of males with a disrupted testicular integrity without a TGCT may contain miR-371a-3p levels above threshold, of which the cellular origin is unknown. OBJECTIVES: Therefore, a series of relevant tissues (frozen and formalin-fixed paraffin-embedded (FFPE), when available) from the complete male urogenital tract (i.e., kidney to urethra and testis to urethra) and semen was investigated for miR-371a-3p levels using targeted quantitative RT-PCR (qRT-PCR). MATERIALS AND METHODS: In total, semen of males with normospermia (n = 11) and oligospermia (n = 3) was investigated, as well as 88 samples derived from 32 different patients. The samples represented one set of tissues related to the entire male urogenital tract (11 anatomical locations), three sets for 10 locations, and four sets for six locations. RESULTS: All testis parenchyma (n = 17) cases showed low miR-371a-3p levels. Eight out of 14 (57%) semen samples showed detectable miR-371a-3p levels, irrespective of the amount of motile spermatozoa, but related to sperm concentration and matched Johnsen score (Spearman's rho correlation coefficient 0.849 and 0.871, p = 0.000, respectively). In all other tissues investigated, miR-371a-3p could not be detected. DISCUSSION: This study demonstrates that the miR-371a-3p in healthy adult males is solely derived from the germ cell compartment. CONCLUSIONS: The observation is important in the context of applying miR-371a-3p as molecular liquid biopsy biomarker for diagnosis and follow-up of patients with malignant (T)GCT. Moreover, miR-371a-3p might be an informative seminal biomarker for testicular germ cell composition.

Malignant testicular germ cell tumors in postpubertal individuals with androgen insensitivity: prevalence, pathology and relevance of single nucleotide polymorphism-based susceptibility profiling.

STUDY QUESTION: What is the prevalence of malignant testicular germ cell tumors (TGCT) and its precursors, (pre-) germ cell neoplasia in situ (GCNIS), in late teenagers and adults who have androgen insensitivity syndrome (AIS) and the impact of an individual's genetic susceptibility to development of TGCT? SUMMARY ANSWER: No GCNIS or TGCT was diagnosed, but pre-GCNIS was identified in 14 and 10% of complete and partial AIS patients, respectively, and was associated with a higher genetic susceptibility score (GSS), with special attention for KITLG (rs995030) and ATFZIP (rs2900333). WHAT IS KNOWN ALREADY: Many adult women with AIS decline prophylactic gonadectomy, while data regarding the incidence, pathophysiology and outcomes of TGCT in postpubertal individuals with AIS are lacking. The relevance of genetic factors, such as single nucleotide polymorphisms (SNPs), in predisposing AIS individuals to TGCT is unknown. STUDY DESIGN, SIZE, DURATION: This multicenter collaborative study on prophylactically removed gonadal tissue was conducted in a pathology lab specialized in germ cell tumor biology. PARTICIPANTS/MATERIALS, SETTING, METHODS: Material from 52 postpubertal individuals with molecularly confirmed AIS (97 gonadal samples) was included; the median age at surgery was 17.5 (14-54) years. Immunohistochemical studies and high-throughput profiling of 14 TGCT-associated SNPs were performed. The main outcome measures were the prevalence of pre-GCNIS, GCNIS and TGCT, and its correlation with a GSS, developed based on the results of recent genome-wide association studies. MAIN RESULTS AND ROLE OF CHANCE: The earliest recognizable change preceding GCNIS, referred to as pre-GCNIS, was present in 14% of individuals with complete and 10% of those with partial AIS at a median age of 16 years. No GCNIS or invasive TGCT were found. The median GSS was significantly greater for those with, compared to those without, pre-GCNIS (P = 0.01), with an overlap between groups. Our data suggest important roles for risk alleles G at KITLG (rs995030) and C at ATFZIP (rs2900333), among the 14 studied TGCT-associated SNPs. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: A limited number of cases were included. WIDER IMPLICATIONS OF THE FINDINGS: Our data suggest that the prevalence of pre-GCNIS in individuals with AIS beyond puberty is around 15%. Genetic susceptibility likely contributes to pre-GCNIS development in AIS but factors related to malignant progression remain unclear. Although data in older patients remain scarce, malignant progression appears to be a rare event, although the natural history of the premalignant lesion remains unknown. Therefore, the practice of routine prophylactic gonadectomy in adults with AIS appears questionable and the patient's preference, after having been fully informed, should be decisive in this matter. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by research grants from the Research Foundation Flanders (FWO) (to M.C.), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq G0D6713N) (to B.B.M. and M.C.) and the European Society for Pediatric Endocrinology (ESPE), granted by Novo Nordisk AB (to J.K.). There are no competing interests.

Adrenal Cushing's syndrome during pregnancy.

Cushing syndrome (CS) during pregnancy is a rare condition with only a few cases reported in the literature. Misdiagnosis of CS is common because of overlapping features like fatigue, weight gain, striae and emotional changes that can occur during normal pregnancy. Changes in maternal hormones and their binding proteins complicate assessment of glucocorticoid hormone levels during gestation. CS during pregnancy is most frequently due to an adrenal adenoma and to a lesser degree to adrenocorticotropic hormone (ACTH) hypersecretion by a pituitary adenoma. Furthermore, aberrant expression of luteinizing hormone (LH) receptors in the adrenal cortex has been suggested to be involved in the pathogenesis of adrenal CS during pregnancy. We report three pregnant women with ACTH-independent Cushing's syndrome and an adrenal tumor. After uncomplicated delivery, patient 1 underwent in vivo testing for aberrant hormone receptor expression by the adenoma. Cortisol responses were found after administration of luteinizing hormone-releasing hormone (LHRH), human chorionic gonadotropin (hCG), glucagon, vasopressin and a standard mixed meal. All patients were treated with laparoscopic adrenalectomy. Adrenal tumor tissue of two patients showed positive immunohistochemical staining of LH receptors. Considering the cortisol responses to LHRH and hCG, and the development of CS during pregnancy in these patients, it is likely that ACTH-independent hypercortisolism was induced by the pregnancy-associated rise in hCG levels that activated aberrantly expressed LH receptors in the adrenal adenoma. Remarkably, adrenal adenomas may simultaneously express multiple aberrant receptors and individual ligands may play a role in the regulation of cortisol production in CS during pregnancy.

Loss of SRY-box2 (SOX2) expression and its impact on survival of patients with oesophageal adenocarcinoma.

BACKGROUND: Oesophageal adenocarcinoma (OAC) is a highly aggressive malignancy with poor survival, which is highly variable amongst patients with comparable conventional prognosticators. Therefore molecular biomarkers are urgently needed to improve the prediction of survival in these patients. SRY (sex determining region Y)-box 2, also known as SOX2, is a transcription factor involved in embryonal development of the gastrointestinal tract as well as in carcinogenesis. The purpose of this study was to see whether SOX2 expression is associated with survival in patients with OAC. METHODS: SOX2 was studied by immunohistochemistry in patients who had undergone potentially curative oesophagectomy for adenocarcinoma. Protein expression of SOX2 was evaluated using tissue microarrays from resection specimens, and results were analysed in relation to the clinical data by Cox regression analysis. SOX2 was evaluated in two independent OAC cohorts (Rotterdam cohort and a multicentre UK cohort). RESULTS: Loss of SOX2 expression was independently predictive of adverse overall survival in the multivariable analysis, adjusted for known factors influencing survival, in both cohorts (Rotterdam cohort: hazard ratio (HR) 1·42, 95 per cent c.i. 1·07 to 1·89, P = 0·016; UK cohort: HR 1·54, 1·08 to 2·19, P = 0·017). When combined with clinicopathological staging, loss of SOX2 showed an increased effect in patients with pT1-2 tumours (P = 0·010) and node-negative OAC (P = 0·038), with an incrementally adverse effect on overall survival for stage I OAC with SOX2 loss (HR 3·18, 1·18 to 8·56; P = 0·022). CONCLUSION: SOX2 is an independent prognostic factor for long-term survival in OAC, especially in patients with stage I OAC.

Gonadal dysgenesis in disorders of sex development: Diagnosis and surgical management.

Recent studies on gonadal histology have improved the understanding of germ cell malignancy risk in patients with disorders of sex development (DSD), and evidence-based gonadal management strategies are gradually emerging. Especially in 46,XY DSD and 45,X/46,XY DSD, which are characterized by gonadal dysgenesis, the risk of germ cell malignancy is significantly increased. This paper summarized the progress over the past 10 years in malignancy risk assessment in patients with DSD, and its implications for optimal surgical handling of the involved gonads.

A MicroRNA Panel in Pancreatic Cyst Fluid for the Risk Stratification of Pancreatic Cysts in a Prospective Cohort.

A subset of pancreatic cystic neoplasms are regarded as precursor lesions of pancreatic cancer, but only a minority of all pancreatic cystic neoplasms will undergo malignant transformation. MicroRNAs are increasingly recognized as molecular targets in carcinogenesis. Previously, a 9-microRNA (miR) signature was suggested to discriminate between high risk and low risk pancreatic cystic neoplasm. In this study, we aimed to validate this 9-miR panel in a prospective cohort. Total miR was isolated from pancreatic cyst fluid and expression of miR18a, miR24, miR30a-3p, miR92a, miR99b, miR106b, miR142-3p, miR342-3p, and miR532-3p was analyzed by singleplex Taqman MicroRNA Assay. A total of 62 patient samples were analyzed. During follow-up, 24 (38.7%) patients underwent resection, of which 6 (9.7%) patients showed at least high grade dysplasia. A logistic regression model presented a "predicted risk" score which significantly differed between low and high risk cysts, either including all patients or only those with histological confirmation of diagnosis. Using a set cut-off of 50%, the sensitivity of the model for the total cohort was 10.0%, specificity 100.0%, positive predicted value 100.0%, negative predicted value 85.2%, and diagnostic accuracy of 85.5%. Thus, while observing a significant difference between low and high risk cysts, clinical implementation of this biomarker panel is as yet unlikely to be beneficial in the management of pancreatic cysts.

Testes in infants with Prader-Willi syndrome: human chorionic gonadotropin treatment, surgery and histology.

PURPOSE: Boys with Prader-Willi syndrome often have undescended testes. Prospective studies on the treatment of cryptorchidism in these patients are lacking. We evaluated the effects of human chorionic gonadotropin administration on testis position in 16 males with Prader-Willi syndrome. In patients who subsequently underwent orchiopexy biopsy was taken and testis histology was evaluated. MATERIALS AND METHODS: A total of 16 boys (median age 1.6 years, IQR 1.2 to 1.8) with Prader-Willi syndrome and cryptorchidism underwent human chorionic gonadotropin stimulation test. After a positive test human chorionic gonadotropin treatment was initiated. Patients received 250 to 500 IU (depending on age) intramuscularly twice weekly for 6 weeks. RESULTS: We found 1 testis in a stable scrotal position, 1 vanished testis and 1 atrophic abdominal testis. Of 29 testes responding to human chorionic gonadotropin 23% reached a stable scrotal position, 62% reached a lower position and 14% did not change position. Thus, 22 testes required orchiopexy. Of 17 obtained biopsies in 12 patients 2 showed germ cells in more than 60% of seminiferous tubules, 3 in 30% to 60% and 7 in less than 30%. In addition, 4 boys had Sertoli cell only syndrome and 1 had a vanished testis. In patients undergoing orchiopexy younger age and increased inhibin B and testosterone levels after human chorionic gonadotropin stimulation were associated with a greater number of germ cell containing tubules. CONCLUSIONS: Human chorionic gonadotropin administration resulted in an anatomically lower testis position in most of our patients with Prader-Willi syndrome, and 23% of testes reached a stable scrotal position. Of the cases 76% required orchiopexy to ensure a stable position in the scrotum.

Pediatric germ cell tumors presenting beyond childhood?

Four cases are reported meeting the criteria of a pediatric (i.e., Type I) testicular germ cell tumor (TGCT), apart from the age of presentation, which is beyond childhood. The tumors encompass the full spectrum of histologies of pediatric TGCT: teratoma, yolk sac tumor, and various combinations of the two, and lack intratubular germ cell neoplasia/carcinoma in situ in the adjacent parenchyma. The neoplasms are (near)diploid, and lack gain of 12p, typical for seminomas and non-seminomas of the testis of adolescents and adults (i.e., Type II). It is proposed that these neoplasms are therefore late appearing pediatric (Type I) TGCT. The present report broadens the concept of earlier reported benign teratomas of the post-pubertal testis to the full spectrum of pediatric TGCT. The possible wide age range of pediatric TGCT, demonstrated in this study, lends credence to the concept that TGCT should according to their pathogenesis be classified into the previously proposed types. This classification is clinically relevant, because Type I mature teratomas are benign tumors, which are candidates for testis conserving surgery, as opposed to Type II mature teratomas, which have to be treated as Type II (malignant) non-seminomas.

Identification of known and novel germ cell cancer-specific (embryonic) miRs in serum by high-throughput profiling.

microRNAs (miRs) are short non-coding RNA molecules (≈21 nucleotides) involved in regulation of translation. As such they are crucial for normal cell development and differentiation as well as cellular maintenance. Dysregulation of miRs has been reported in various diseases, including cancer. Interestingly, miRs can be informative as tumor classifiers and disease biomarkers. Recent studies demonstrated the presence of miRs in body fluids like serum, thus providing a putative non-invasive tool to study and monitor disease state. Earlier targeted studies by several independent groups identified specific embryonic miRs as characteristic for germ cell tumors (GCT) (miR-371-2-3 & miR-302/367 clusters). This study reports a high-throughput miR profiling (≈750 miRs) approach on serum from testicular germ cell tumor patients (14 seminoma and 10 non-seminoma) and controls (n = 11), aiming at independent identification of miRs as candidate biomarkers for testicular GCT. A magnetic bead capture system was used to isolate miRs from serum. Subsequently, the TaqMan Array Card 3.0 platform was used for profiling. The previously identified miRs 371 and 372 were confirmed to be specifically elevated in serum from germ cell tumor patients. In addition, several novels miRs were identified that were discriminative between germ cell cancer and controls: miR-511, -26b, -769, -23a, -106b, -365, -598, -340, and let-7a. In conclusion, this study validates the power of the embryonic miRs 371 and 372 in detecting malignant GCT (SE and NS) based on serum miR levels and identifies several potential novel miR targets.

45,X/46,X,psu dic(Y) gonadal dysgenesis: influence of the two cell lines on the clinical phenotype, including gonadal histology.

A child born with ambiguous genitalia (Prader III) was found to have a 45,X[92.2%]/46,X,psu dic(Y)(p12)[7.8%] karyotype in peripheral blood lymphocytes. The testosterone level was consistent with that of a normal male; however, gonadotropins were elevated. Ultrasound and endoscopy of the urogenital sinus revealed well-developed Müllerian structures. At 3.5 months, the child was operated for right-sided incarcerated hernia, and the gonad situated at the inguinal region was biopsied and classified as primitive testis. Based on the presence of Müllerian structures, anatomy of external genitalia and wish of the parents, the child was assigned female gender. She underwent removal of the left gonad at 4 months during another acute surgery; histology was similar to the right gonad. The rest of the right gonad was removed at 16 months, and feminizing genitoplasty took place at 3 years. The right and left gonad contained 28 and 22% of cells with a Y chromosome, respectively. During further histological examination, dysgenetic features of the gonads were discovered. Some germ cells displayed abnormal development based on the specific expression of immunohistochemical markers (OCT3/4, TSPY, KITLG), indicating a possible risk for future malignant germ cell tumor development. Contribution of the 45,X cell line to the phenotype was also observed: the patient developed celiac disease, and her growth pattern resembled that of Turner syndrome responding to growth hormone treatment.

Maintaining success, reducing treatment burden, focusing on survivorship: highlights from the third European consensus conference on diagnosis and treatment of germ-cell cancer.

In November 2011, the Third European Consensus Conference on Diagnosis and Treatment of Germ-Cell Cancer (GCC) was held in Berlin, Germany. This third conference followed similar meetings in 2003 (Essen, Germany) and 2006 (Amsterdam, The Netherlands) [Schmoll H-J, Souchon R, Krege S et al. European consensus on diagnosis and treatment of germ-cell cancer: a report of the European Germ-Cell Cancer Consensus Group (EGCCCG). Ann Oncol 2004; 15: 1377-1399; Krege S, Beyer J, Souchon R et al. European consensus conference on diagnosis and treatment of germ-cell cancer: a report of the second meeting of the European Germ-Cell Cancer Consensus group (EGCCCG): part I. Eur Urol 2008; 53: 478-496; Krege S, Beyer J, Souchon R et al. European consensus conference on diagnosis and treatment of germ-cell cancer: a report of the second meeting of the European Germ-Cell Cancer Consensus group (EGCCCG): part II. Eur Urol 2008; 53: 497-513]. A panel of 56 of 60 invited GCC experts from all across Europe discussed all aspects on diagnosis and treatment of GCC, with a particular focus on acute and late toxic effects as well as on survivorship issues. The panel consisted of oncologists, urologic surgeons, radiooncologists, pathologists and basic scientists, who are all actively involved in care of GCC patients. Panelists were chosen based on the publication activity in recent years. Before the meeting, panelists were asked to review the literature published since 2006 in 20 major areas concerning all aspects of diagnosis, treatment and follow-up of GCC patients, and to prepare an updated version of the previous recommendations to be discussed at the conference. In addition, ∼50 E-vote questions were drafted and presented at the conference to address the most controversial areas for a poll of expert opinions. Here, we present the main recommendations and controversies of this meeting. The votes of the panelists are added as online supplements.

Genetic analysis of NR0B1 in congenital adrenal hypoplasia patients: identification of a rare regulatory variant resulting in congenital adrenal hypoplasia and hypogonadal hypogonadism without testicular carcinoma in situ.

There have been few testicular histology reports of adult patients with congenital adrenal hypoplasia/hypogonadal hypogonadism (AHC/HH), but Leydig cell hyperplasia has been observed, an indicator of the possibility of malignant transformation. We aimed to define the basis of AHC/HH in 4 pedigrees of different ethnic backgrounds. One patient was elected to have testicular biopsy which was examined for evidence of carcinoma in situ (CIS). NR0B1 mutation analysis was performed by sequence analysis. NR0B1 expression was investigated by RT-PCR. Testicular biopsy sections were stained with HE or immunostained for OCT3/4, an established marker of CIS. We identified NR0B1 variants in the 4 AHC pedigrees: pedigree 1 (United Arab Emirates), c.1130A>G predicting p.(Glu377Gly); pedigree 2 (English Caucasian), c.327C>A predicting p.(Cys109*); pedigree 3 (Oman), a 6-bp deletion of a direct repeat, c.857_862delTGGTGC predicting p.(Leu286_Val287del); pedigree 4 (English Caucasian), c.1168+1G>A, a regulatory variant within the NR0B1 splice donor site. This last male patient, aged 30 years, presented with evidence of HH but incomplete gonadotrophin deficiency, following an earlier diagnosis of Addison's disease at 3 years. Hormonal therapy induced virilisation. Testicular biopsy was performed. The c.1168+1G>A variant abrogated normal splicing of testicular mRNA. Histological examination showed poorly organised testicular architecture and absence of spermatozoa. Morphological analyses and the absence of immunohistochemical staining for OCT3/4 excluded the presence of malignant germ cell cancer and its precursor lesion, CIS. These studies add to the knowledge of the types and ethnic diversity of NR0B1 mutations and their associated phenotypes, and provide insight into the assessment and interpretation of testicular histology in AHC and HH.

A novel AMH missense mutation in a patient with persistent Müllerian duct syndrome.

Persistent Müllerian duct syndrome (PMDS) is characterized by the presence of a uterus, fallopian tubes, and the upper part of the vagina in phenotypic normal male patients. Here, we report a patient diagnosed with PMDS with a novel homozygous missense mutation in the anti-Müllerian hormone (AMH) gene (single nucleotide insertion (C) at position 208 (c.208dup, p.Leu70fs)) leading to a frameshift and the introduction of a premature stop codon. Biopsy of both gonads revealed that germ cells were present in an irregular distribution. However, the absence of OCT3/4, PLAP and c-KIT expression indicated physiological maturation.

Pubertal androgenization and gonadal histology in two 46,XY adolescents with NR5A1 mutations and predominantly female phenotype at birth.

OBJECTIVE: Most patients with NR5A1 (SF-1) mutations and poor virilization at birth are sex-assigned female and receive early gonadectomy. Although studies in pituitary-specific Sf-1 knockout mice suggest hypogonadotropic hypogonadism, little is known about endocrine function at puberty and on germ cell tumor risk in patients with SF-1 mutations. This study reports on the natural course during puberty and on gonadal histology in two adolescents with SF-1 mutations and predominantly female phenotype at birth. DESIGN AND METHODS: Clinical and hormonal data and histopathological studies are reported in one male and one female adolescent with, respectively, a nonsense mutation (c.9T>A, p.Tyr3X) and a deletion of the first two coding exons (NCBI36/hg18 Chr9:g.(126306276-126307705)_(126303229-126302828)del) of NR5A1, both predicted to fully disrupt gene function. RESULTS: LH and testosterone concentrations were in the normal male range, virilization was disproportionate to the neonatal phenotype. In the girl, gonadectomy at 13 years revealed incomplete spermatogenesis and bilateral precursor lesions of testicular carcinoma in situ. In the boy, at the age of 12, numerous germ cells without signs of malignancy were present in bilateral testicular biopsy specimen. CONCLUSIONS: In SF-1 mutations, the neonatal phenotype poorly predicts virilization at puberty. Even in poorly virilized cases at birth, male gender assignment may allow spontaneous puberty without signs of hypogonadotropic hypogonadism, and possibly fertility. Patients with SF-1 mutations are at increased risk for malignant germ cell tumors. In case of preserved gonads, early orchidopexy and germ cell tumor screening is warranted. The finding of premalignant and/or malignant changes should prompt gonadectomy or possibly irradiation.

DNA methylation profiles delineate epigenetic heterogeneity in seminoma and non-seminoma.

BACKGROUND: It remains important to understand the biology and identify biomarkers for less studied cancers like testicular cancer. The purpose of this study was to determine the methylation frequency of several cancer-related genes in different histological types of testicular cancer and normal testis tissues (NT). METHODS: DNA was isolated from 43 seminomas (SEs), 14 non-SEs (NSEs) and 23 NT, and was assayed for promoter methylation status of 15 genes by quantitative methylation-specific PCR. The methylation status was evaluated for an association with cancer, and between SEs and NSEs. RESULTS: We found differential methylation pattern in SEs and NSEs. MGMT, VGF, ER-ß and FKBP4 were predominately methylated in NSEs compared with SEs. APC and hMLH1 are shown to be significantly more methylated in both subtypes in comparison with NT. When combining APC, hMLH1, ER-ß and FKBP4, it is possible to identify 86% of the NSEs, whereas only 7% of the SEs. CONCLUSIONS: Our results indicate that the methylation profile of cancer-associated genes in testicular cancer correlates with histological types and show cancer-specific pattern for certain genes. Further methylation analysis, in a larger cohort is needed to elucidate their role in testicular cancer development and potential for therapy, early detection and disease monitoring.

Specific detection of OCT3/4 isoform A/B/B1 expression in solid (germ cell) tumours and cell lines: confirmation of OCT3/4 specificity for germ cell tumours.

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.

Gonadal development and tumor formation at the crossroads of male and female sex determination.

Malignant germ cell tumor (GCT) formation is a well-known complication in the management of patients with a disorder of sex development (DSD). DSDs are defined as congenital conditions in which development of chromosomal, gonadal, or anatomical sex is atypical. DSD patients in whom the karyotype - at least at the gonadal level - contains (a part of) the Y chromosome are at increased risk for neoplastic transformation of germ cells, leading to the development of the so-called 'type II germ cell tumors'. However, tumor risk in the various forms of DSD varies considerably between the different diagnostic groups. This contribution integrates our actual knowledge on the pathophysiology of tumor development in DSDs, recent findings on gonadal (mal)development in DSD patients, and possible correlations between the patient's phenotype and his/her risk for germ cell tumor development.