Intratubular germ cell neoplasia of the human testis: heterogeneous protein expression and relation to invasive potential.

Testicular germ cell cancer develops from premalignant intratubular germ cell neoplasia, unclassified cells that are believed to arise from failure of normal maturation of fetal germ cells from gonocytes (OCT4(+)/MAGEA4(-)) into pre-spermatogonia (OCT4(-)/MAGEA4(+)). Intratubular germ cell neoplasia cell subpopulations based on stage of germ cell differentiation have been described, however the importance of these subpopulations in terms of invasive potential has not been reported. We hypothesized that cells expressing an immature (OCT4(+)/MAGEA4(-)) germ cell profile would exhibit an increased proliferation rate compared with those with a mature profile (OCT4(+)/MAGEA4(+)). Therefore, we performed triple immunofluorescence and stereology to quantify the different intratubular germ cell neoplasia cell subpopulations, based on expression of germ cell (OCT4, PLAP, AP2γ, MAGEA4, VASA) and proliferation (Ki67) markers, in testis sections from patients with preinvasive disease, seminoma, and non-seminoma. We compared these subpopulations with normal human fetal testis and with seminoma cells. Heterogeneity of protein expression was demonstrated in intratubular germ cell neoplasia cells with respect to gonocyte and spermatogonial markers. It included an embryonic/fetal germ cell subpopulation lacking expression of the definitive intratubular germ cell neoplasia marker OCT4, that did not correspond to a physiological (fetal) germ cell subpopulation. OCT4(+)/MAGEA4(-) cells showed a significantly increased rate of proliferation compared with the OCT4(+)/MAGEA4(+) population (12.8 versus 3.4%, P<0.0001) irrespective of histological tumor type, reflected in the predominance of OCT4(+)/MAGEA4(-) cells in the invasive tumor component. Surprisingly, OCT4(+)/MAGEA4(-) cells in patients with preinvasive disease showed significantly higher proliferation compared to those with seminoma or non-seminoma (18.1 versus 10.2 versus 7.2%, P<0.05, respectively). In conclusion, this study has demonstrated that OCT4(+)/MAGEA4(-) cells are the most frequent and most proliferative cell population in tubules containing intratubular germ cell neoplasia, which appears to be an important factor in determining invasive potential of intratubular germ cell neoplasia to seminomas.

Genome-wide methylation analysis in patients with proximal hypospadias - a pilot study and review of the literature.

In patients with proximal hypospadias, often no genetic cause is identified despite extensive genetic testing. Many genes involved in sex development encode transcription factors with strict timing and dosing of the gene products. We hypothesised that there might be recurrent differences in DNA methylation in boys with hypospadias and that these might differ between patients born small versus appropriate for gestational age. Genome-wide Methylated DNA sequencing (MeD-seq) was performed on 32bp LpnPI restriction enzyme fragments after RE-digestion in leucocytes from 16 XY boys with unexplained proximal hypospadias, one with an unexplained XX testicular disorder/difference of sex development (DSD) and twelve, healthy, sex- and age-matched controls. Five of seven differentially methylated regions (DMRs) between patients and XY controls were in the Long Intergenic Non-Protein Coding RNA 665 (LINC00665; CpG24525). Three patients showed hypermethylation of MAP3K1. Finally, no DMRs in XX testicular DSD associated genes were identified in the XX boy versus XX controls. In conclusion, we observed no recognizable epigenetic signature in 16 boys with XY proximal hypospadias and no difference between children born small versus appropriate for gestational age. Comparison to previous methylation studies in individuals with hypospadias did not show consistent findings, possibly due to the use of different inclusion criteria, tissues and methods.

SRY mutation analysis by next generation (deep) sequencing in a cohort of chromosomal Disorders of Sex Development (DSD) patients with a mosaic karyotype.

BACKGROUND: The presence of the Y-chromosome or Y chromosome-derived material is seen in 4-60% of Turner syndrome patients (Chromosomal Disorders of Sex Development (DSD)). DSD patients with specific Y-chromosomal material in their karyotype, the GonadoBlastoma on the Y-chromosome (GBY) region, have an increased risk of developing type II germ cell tumors/cancer (GCC), most likely related to TSPY. The Sex determining Region on the Y gene (SRY) is located on the short arm of the Y-chromosome and is the crucial switch that initiates testis determination and subsequent male development. Mutations in this gene are responsible for sex reversal in approximately 10-15% of 46,XY pure gonadal dysgenesis (46,XY DSD) cases. The majority of the mutations described are located in the central HMG domain, which is involved in the binding and bending of the DNA and harbors two nuclear localization signals. SRY mutations have also been found in a small number of patients with a 45,X/46,XY karyotype and might play a role in the maldevelopment of the gonads. METHODS: To thoroughly investigate the presence of possible SRY gene mutations in mosaic DSD patients, we performed next generation (deep) sequencing on the genomic DNA of fourteen independent patients (twelve 45,X/46,XY, one 45,X/46,XX/46,XY, and one 46,XX/46,XY). RESULTS AND CONCLUSIONS: The results demonstrate that aberrations in SRY are rare in mosaic DSD patients and therefore do not play a significant role in the etiology of the disease.

Spermatocytic seminoma: toward further understanding of pathogenesis.

Human germ cell tumours comprise a heterogeneous group of neoplasms which, based on pathobiological, genetic and clinical characteristics, can be subdivided into different entities. One of these subgroups relates to the so-called spermatocytic seminomas, benign tumours only found in the testis, preferentially in elderly men. Various developmental models for this type of germ cell tumour have been proposed and it is clear that spermatocytic seminoma has a pathogenesis independent from that of seminoma. A recent study examining expression of spermatogonial markers shows that spermatocytic seminomas are a heterogeneous group of tumours, with a supposed difference in origin, ie the majority from A(pale) or B spermatogonia, and a minority from A(dark) spermatogonia. However, this does not exclude an earlier cell of origin, possibly explaining the unique properties of this type of human germ cell tumour, with various counterparts in animals.

Tumor risk and clinical follow-up in patients with disorders of sex development.

A subset of patients with disorders of sex development (DSD) is at risk for malignant germ cell tumors (GCTs). The degree of gonadal differentiation (or "testicularization" in the presence of a specific part of the Y chromosome), in combination with expression of embryonic germ cell markers, and (a) Y specific gene(s) related to cell-cycle control and proliferation, determines this risk. Incompletely matured Sertoli/granulosa cells are insufficiently capable of directing the normal mitotic block/meiotic induction germ cell program, and as a result, embryonic germ cells are delayed or blocked in their normal maturation process. Thereby, they remain pluripotent and gain increased mitotic and survival characteristics, being the first step in the pathogenesis of GCTs. The patient's underlying genetic defect and phenotype might be informative in assessing the degree of gonadal "testicularization" on a clinical basis. Current knowledge allows development of an informative cancer risk assessment of DSD patients.

Mediastinal germ cell tumors: many questions and perhaps an answer.

Some germ cell tumors (GCTs) in men develop into hematologic malignancies; however, the clonal origins of such malignancies remain unknown. In this issue of the JCI, Taylor, Donoghue, et al. unravel the clonal relationship between primary mediastinal nonseminomas (PMNs) and hematologic somatic-type malignancies (HSTMs). Whole-exome sequencing was used to construct phylogenetic trees of the PMNs and the ensuing HSTM clones. HSTMs were derived from multiple distinct clones not detected within the PMNs. Clones from PMNs and HSTMs shared a common precursor, arguably an embryonal carcinoma cell resulting from a reprogrammed primordial germ cell from the thymus. Mutational and copy number variation analysis of a large cohort of patients with PMNs also demonstrated a high prevalence of TP53 mutations not found in testicular nonseminomas. These data likely explain why patients with PMNs are frequently resistant to platinum-based chemotherapy and provide TP53 mutations as potential targets.

Effect of neoadjuvant chemoradiotherapy on p53 and SOX2 protein expression in esophageal adenocarcinoma.

Aim: To determine if neoadjuvant chemoradiotherapy (nCRT) affects p53 and SOX2 expression in esophageal adenocarcinoma (EAC). Materials & methods: Comparison of p53 and SOX2 expression in 100 paired pre- and post-nCRT EAC samples. Results: Aberrant p53 was largely concordant (75/83, 90%), while 13/18 (72%) pre-nCRT samples with wild-type (WT) p53 staining, showed aberrant staining in paired post-nCRT samples. Similarly, 31/45 (69%) with previous WT SOX2 showed SOX2 loss in paired post-nCRT samples, whereas aberrant SOX2 loss was concordant in 50/55 (91%) cases. The prognostic values of both markers regarding survival differ before and after nCRT. Conclusion: Aberrant expression of p53 and SOX2 staining in EAC tissue is unaffected by nCRT. Conversely, the WT-staining pattern frequently changed to aberrant expression.

Malignant recurrence after mature Sacrococcygeal teratoma: A meta-analysis and review of the literature.

BACKGROUND AND AIMS: Sacrococcygeal teratoma (SCT) is a rare extragonadal germ cell tumour mostly diagnosed during infancy and early childhood. Neonatal SCTs are mostly mature, but can also contain immature and/or malignant components. Recurrence of an SCT alters prognosis, especially when it is malignant, of which its mechanism is not yet fully understood. This study is a review and meta-analysis of the literature on malignant recurrences after an initially mature SCT. METHODS: A literature search was performed to identify studies describing children with SCT and presenting specific information on histology of the initial tumour as well as the recurrence. Random effect models for mature recurrence and malignant recurrence after an initially mature SCT were employed to pool study-specific percentages in order to estimate an overall percentage and its associated 95 % confidence intervals (CI). Inverse variance method, which gives more weight to larger studies, was used to pool outcomes for the different studies. RESULTS: A total of 22 articles, comprising 1516 patients with SCT, were included in the meta-analysis. The pooled proportions of mature and malignant recurrences after mature SCT were 3 % (95 % CI 1-4 %) and 5% (95 % CI 3-6 %), respectively. Fifty-seven (56 %) of a total of 102 recurrences after resection of an initially mature SCT were malignant, mostly yolk sac tumour (YST). Many recurrences occurred within 1-6 years, however some occurred as long as 20 years after initial diagnosis. CONCLUSIONS: A substantial number of recurrences of mature SCT present as a malignant tumour. Overlooking malignant components on initial pathological evaluation and the progression of mature SCT cells to malignant cells may play a role. Treatment of mature SCTs with resection alone requires thorough follow-up of at least 6 years. Future research is needed to determine whether SCTs with malignant microfoci should be treated or followed-up differently from mature or immature SCTs. In addition, the value of serum biomarkers in follow-up after SCT needs to be further evaluated.

Differential expression of DNA methyltransferases and demethylases among the various testicular germ cell tumor subtypes.

Aim: Characterize DNA methyltransferases/demethylases expression in testicular germ cell tumors (TGCTs). Methods:In silico analysis of TCGA database, assessment of transcript levels of most relevant enzymes in four TGCT cell lines and validation in patient cohort (real-time quantitative polymerase chain reaction; immunohistochemistry). Results:DNMT3A, DNMT3B and TET2 were the most differentially expressed between seminomas (SEs) and nonseminomas (NSs). DNMT3B was significantly overexpressed in NS-related cell lines, and the opposite was found for TET2. Significantly higher DNMT3A/B mRNA expression was observed in NS, indicating a role for de novo methylation in reprogramming. Significantly higher TET2 protein expression was observed in SEs, suggesting active demethylation contributes for SE hypomethylated state. More differentiated histologies disclosed distinct expression patterns. Conclusion: DNA-modifying enzymes are differentially expressed between TGCT subtypes, influencing reprogramming and differentiation.

Efficacy of HDAC Inhibitors Belinostat and Panobinostat against Cisplatin-Sensitive and Cisplatin-Resistant Testicular Germ Cell Tumors.

Novel treatment options are needed for testicular germ cell tumor (TGCT) patients, particularly important for those showing or developing cisplatin resistance, the major cause of cancer-related deaths. As TGCTs pathobiology is highly related to epigenetic (de)regulation, epidrugs are potentially effective therapies. Hence, we sought to explore, for the first time, the effect of the two most recently FDA-approved HDAC inhibitors (HDACis), belinostat and panobinostat, in (T)GCT cell lines including those resistant to cisplatin. In silico results were validated in 261 patient samples and differential expression of HDACs was also observed across cell lines. Belinostat and panobinostat reduced cell viability in both cisplatin-sensitive cells (NCCIT-P, 2102Ep-P, and NT2-P) and, importantly, also in matched cisplatin-resistant subclones (NCCIT-R, 2102Ep-R, and NT2-R), with IC50s in the low nanomolar range for all cell lines. Treatment of NCCIT-R with both drugs increased acetylation, induced cell cycle arrest, reduced proliferation, decreased Ki67 index, and increased p21, while increasing cell death by apoptosis, with upregulation of cleaved caspase 3. These findings support the effectiveness of HDACis for treating TGCT patients in general, including those developing cisplatin resistance. Future studies should explore them as single or combination agents.

Do pathologists agree with each other on the histological assessment of pT1b oesophageal adenocarcinoma?

Background: In early (T1) oesophageal adenocarcinoma (OAC), the histological profile of an endoscopic resection specimen plays a pivotal role in the prediction of lymph node metastasis and the potential need for oesophagectomy with lymphadenectomy. Objective: To evaluate the inter-observer agreement of the histological assessment of submucosal (pT1b) OAC. Methods: Surgical and endoscopic resection specimens with pT1b OAC were independently reviewed by three gastrointestinal pathologists. Agreement was determined by intraclass correlation coefficient for continuous variables, and Fleiss' kappa (κ) for categorical variables. Bland-Altman plots of the submucosal invasion depth were made. Results: Eighty-five resection specimens with pT1b OAC were evaluated. The agreement was good for differentiation grade (κ=0.77, 95% confidence interval (CI) 0.68-0.87), excellent for lymphovascular invasion (κ=0.88, 95% CI 0.76-1.00) and moderate for submucosal invasion depth using the Paris and Pragmatic classifications (κ=0.60, 95% CI 0.49-0.72 and κ=0.42, 95% CI 0.33-0.51, respectively). Systematic mean differences between pathologists were detected for the measurement of submucosal invasion depth, ranging from 297 µm to 602 µm. Conclusions: A substantial discordance was found between pathologists for the measurement of submucosal invasion depth in pT1b OAC. Differences may lead to an over- or underestimation of the lymph node metastasis risk, with grave implications for the treatment strategy. Review by a second gastrointestinal pathologist is recommended to improve differentiating between a favourable and an unfavourable histological profile.

Etiology and early pathogenesis of malignant testicular germ cell tumors: towards possibilities for preinvasive diagnosis.

Malignant testicular germ cell tumors (TGCT) are the most frequent cancers in Caucasian males (20-40 years) with an 70% increasing incidence the last 20 years, probably due to combined action of (epi)genetic and (micro)environmental factors. It is expected that TGCT have carcinoma in situ(CIS) as their common precursor, originating from an embryonic germ cell blocked in its maturation process. The overall cure rate of TGCT is more than 90%, however, men surviving TGCT can present long-term side effects of systemic cancer treatment. In contrast, men diagnosed and treated for CIS only continue to live without these long-term side effects. Therefore, early detection of CIS has great health benefits, which will require an informative screening method. This review described the etiology and early pathogenesis of TGCT, as well as the possibilities of early detection and future potential of screening men at risk for TGCT. For screening, a well-defined risk profile based on both genetic and environmental risk factors is needed. Since 2009, several genome wide association studies (GWAS) have been published, reporting on single-nucleotide polymorphisms (SNPs) with significant associations in or near the genes KITLG, SPRY4, BAK1, DMRT1, TERT, ATF7IP, HPGDS, MAD1L1, RFWD3, TEX14, and PPM1E, likely to be related to TGCT development. Prenatal, perinatal, and postnatal environmental factors also influence the onset of CIS. A noninvasive early detection method for CIS would be highly beneficial in a clinical setting, for which specific miRNA detection in semen seems to be very promising. Further research is needed to develop a well-defined TGCT risk profile, based on gene-environment interactions, combined with noninvasive detection method for CIS.