Humanised Mice and Immunodeficient Mice (NSG) Are Equally Sensitive for Prediction of Stem Cell Malignancy in the Teratoma Assay.

The use of human pluripotent stem cells (hPSCs) in regenerative medicine has great potential. However, it is important to exclude that these cells can undergo malignant transformation, which could lead to the development of malignant tumours. This property of hPSCs is currently being tested using the teratoma assay, through which cells are injected into immunodeficient mice. Transplantation of stem cells in immunocompromised recipient animals certainly has a much higher incidence of tumour formation. On the other hand, the results obtained in immunodeficient mice could indicate a risk of tumour formation that is practically not present in the human immunocompetent recipient. The presence of a humanised immune system might be more representative of the human situation; therefore, we investigated if the demonstrated malignant features of chosen and well-characterised stem cell lines could be retrieved and if new features could arise in a humanised mouse model. Hu-CD34NSGTM (HIS) mice were compared side by side with immunocompromised mice (NSG) after injection of a set of benign (LU07) and malignant (LU07+dox and 2102Ep) cell lines. Analysis of the tumour development, histological composition, pathology evaluation, and malignancy-associated miRNA expression levels, both in tumour and plasma samples, revealed no differences among mouse groups. This indicates that the HIS mouse model is comparable to, but not more sensitive than, the NSG immunodeficient model for studying the malignancy of stem cells. Since in vivo teratoma assay is cumbersome, in vitro methods for the detection of malignancy are urgently needed.

The Role of TP53 in Cisplatin Resistance in Mediastinal and Testicular Germ Cell Tumors.

Germ cell tumors (GCTs) are considered to be highly curable; however, there are major differences in the outcomes related to histology and anatomical localization. GCTs originating from the testis are, overall, sensitive to platinum-based chemotherapy, whereas GCTs originating from the mediastinum show a worse response, which remains largely unexplained. Here, we address the differences among GCTs from two different anatomical locations (testicular versus mediastinal/extragonadal), with a specific focus on the role of the P53 pathway. It was recently shown that GCTs with TP53 mutations most often localize to the mediastinum. To elucidate the underlying mechanism, TP53 knock-out lines were generated in cisplatin-sensitive and -resistant clones of the representative 2102Ep cell line (wild-type TP53 testicular GCT) and NCCIT cell line (hemizygously mutated TP53, mutant TP53 mediastinal GCT). The full knock-out of TP53 in 2102Ep and resistant NCCIT resulted in an increase in cisplatin resistance, suggesting a contributing role for P53, even in NCCIT, in which P53 had been reported to be non-functional. In conclusion, these results suggest that TP53 mutations contribute to the cisplatin-resistant phenotype of mediastinal GCTs and, therefore, are a potential candidate for targeted treatment. This knowledge provides a novel model system to elucidate the underlying mechanism of clinical behavior and possible alternative treatment of the TP53 mutant and mediastinal GCTs.

Recurrence of a Mediastinal Germ-Cell Tumor as a Somatic-Type Malignancy: A Complex Case Report.

Background and case: An adolescent male presented with a second mediastinal tumor 1.5 years after treatment of a proven malignant germ-cell tumor in that location. The differential diagnosis included a recurrent germ-cell tumor or a non-germ cell malignancy. Serum tumor markers alpha-fetoprotein (AFP) and human chorionic gonadotrophin (HCG) were negative. The first biopsy was not informative, and the second biopsy gave a broad differential diagnosis including secondary non-germ cell malignancy using histology and immunohistochemistry. DNA methylation profiling, RNA sequencing, and targeted microRNA371a-3p profiling was subsequently performed, without a supportive result. After resection of the tumor the definitive diagnosis yielded two secondary non-germ cell malignancies in the form of a leiomyosarcoma and a solitary neuro endocrine carcinoma (NEC). In spite of the differences between the molecular profiles of the initial germ-cell tumor, the leiomyosarcoma and large-cell NEC are clonally related, as determined by the presence of identical chromosomal breakpoints. The copy number profiles suggest an initial polyploidization step, followed by various independent chromosomal gains and losses. This case demonstrates that germ-cell tumors must be evaluated carefully, including molecularly, in which the non-germ cell malignancy is negative for miR-371a-3p, both in tissue as well as in serum, in contrast to the primary tumor. We conclude that the patient presented with a primary type II mediastinal GCT and, a year and a half later, followed by a leiomyosarcoma and a large-cell NEC presenting as two secondary somatic-type malignancies clonally related to the original GCT. Conclusions: Malignant germ-cell tumors are known to recur as a somatic-type malignancy in very rare cases. This case report illustrates the challenges faced in defining the nature and clonality of the secondary somatic-type malignancies.

Napabucasin overcomes cisplatin resistance in ovarian germ cell tumor-derived cell line by inhibiting cancer stemness.

BACKGROUND: Cisplatin resistance of ovarian yolk sac tumors (oYST) is a clinical challenge due to dismal patient prognosis, even though the disease is extremely rare. We investigated potential association between cisplatin resistance and cancer stem cell (CSC) markers in chemoresistant oYST cells and targeting strategies to overcome resistance in oYST. METHODS: Chemoresistant cells were derived from chemosensitive human oYST cells by cultivation in cisplatin in vitro. Derivative cells were characterized by chemoresistance, functional assays, flow cytometry, gene expression and protein arrays focused on CSC markers. RNAseq, methylation and microRNA profiling were performed. Quail chorioallantoic membranes (CAM) with implanted oYST cells were used to analyze the micro-tumor extent and interconnection with the CAM. Tumorigenicity in vivo was determined on immunodeficient mouse model. Chemoresistant cells were treated by inhibitors intefering with the CSC properties to examine the chemosensitization to cisplatin. RESULTS: Long-term cisplatin exposure resulted in seven-fold higher IC50 value in resistant cells, cross-resistance to oxaliplatin and carboplatin, and increased migratory capacity, invasiveness and tumorigenicity, associated with hypomethylation of differentially methylated genes/promotors. Resistant cells exhibited increased expression of prominin-1 (CD133), ATP binding cassette subfamily G member 2 (ABCG2), aldehyde dehydrogenase 3 isoform A1 (ALDH3A1), correlating with reduced gene and promoter methylation, as well as increased expression of ALDH1A3 and higher overall ALDH enzymatic activity, rendering them cross-resistant to DEAB, disulfiram and napabucasin. Salinomycin and tunicamycin were significantly more toxic to resistant cells. Pretreatment with napabucasin resensitized the cells to cisplatin and reduced their tumorigenicity in vivo. CONCLUSIONS: The novel chemoresistant cells represent unique model of refractory oYST. CSC markers are associated with cisplatin resistance being possible targets in chemorefractory oYST.

Prediction of relapse in stage I testicular germ cell tumor patients on surveillance: investigation of biomarkers.

BACKGROUND: Better biomarkers for assessing risk of relapse in stage I testicular germ cell tumor patients are needed, to complement classical histopathological variables. We aimed to assess the prognostic value of previously suggested biomarkers, related to proliferation (MIB-1 and TEX19) and to immune microenvironment (CXCL12, CXCR4, beta-catenin and MECA-79) in a surveillance cohort of stage I testicular germ cell tumor patients. METHODS: A total of 70 patients were included. Survival analyses were performed, including Cox regression models. RESULTS: Patients with vascular invasion and elevated human chorionic gonadotropin levels showed significantly poorer relapse-free survival in multivariable analysis (hazard ratio = 2.820, 95% confidence interval 1.257-6.328; hazard ratio = 3.025, 95% confidence interval 1.345-6.808). Patients with no vascular invasion but with MIB-1 staining in > 50% tumor cells showed significantly shorter relapse-free survival (p = 0.042). TEX19 nuclear immunoexpression was confirmed in spermatogonial cells, and weak cytoplasmic immunoexpression was depicted in 15/70 tumors, not significantly impacting survival. CXCL12 immunoexpression in tumor cells did not associate with relapse, but non-seminoma patients exhibiting vascular invasion and CXCL12-positive stromal/inflammatory cells showed significantly improved relapse-free survival (p = 0.015). Exclusively nuclear immunoexpression of CXCR4 associated with better relapse-free survival (p = 0.032), but not after adjusting for vascular invasion. Patients with higher beta-catenin scores showed a tendency for poorer relapse-free survival (p = 0.056). MECA-79 immunoexpression was absent. CONCLUSIONS: The informative protein biomarkers (i.e., MIB-1, CXCL12, beta-catenin, and possibly CXCR4) may prove useful for risk-stratifying patients if validated in larger, multicentric and well-defined studies. Currently, classical histopathological features of testicular germ cell tumors remain key for relapse prediction.

Molecular heterogeneity and early metastatic clone selection in testicular germ cell cancer development.

BACKGROUND: Testicular germ cell cancer (TGCC), being the most frequent malignancy in young Caucasian males, is initiated from an embryonic germ cell. This study determines intratumour heterogeneity to unravel tumour progression from initiation until metastasis. METHODS: In total, 42 purified samples of four treatment-resistant nonseminomatous (NS) TGCC were investigated, including the precursor germ cell neoplasia in situ (GCNIS) and metastatic specimens, using whole-genome and targeted sequencing. Their evolution was reconstructed. RESULTS: Intratumour molecular heterogeneity did not correspond to the supposed primary tumour histological evolution. Metastases after systemic treatment could be derived from cancer stem cells not identified in the primary cancer. GCNIS mostly lacked the molecular marks of the primary NS and comprised dominant clones that failed to progress. A BRCA-like mutational signature was observed without evidence for direct involvement of BRCA1 and BRCA2 genes. CONCLUSIONS: Our data strongly support the hypothesis that NS is initiated by whole-genome duplication, followed by chromosome copy number alterations in the cancer stem cell population, and accumulation of low numbers of somatic mutations, even in therapy-resistant cases. These observations of heterogeneity at all stages of tumourigenesis should be considered when treating patients with GCNIS-only disease, or with clinically overt NS.

Imprints and DPPA3 are bypassed during pluripotency- and differentiation-coupled methylation reprogramming in testicular germ cell tumors.

Testicular germ cell tumors (TGCTs) share germline ancestry but diverge phenotypically and clinically as seminoma (SE) and nonseminoma (NSE), the latter including the pluripotent embryonal carcinoma (EC) and its differentiated derivatives, teratoma (TE), yolk sac tumor (YST), and choriocarcinoma. Epigenomes from TGCTs may illuminate reprogramming in both normal development and testicular tumorigenesis. Herein we investigate pure-histological forms of 130 TGCTs for conserved and subtype-specific DNA methylation, including analysis of relatedness to pluripotent stem cell (ESC, iPSC), primordial germ cell (PGC), and differentiated somatic references. Most generally, TGCTs conserve PGC-lineage erasure of maternal and paternal genomic imprints and DPPA3 (also known as STELLA); however, like ESCs, TGCTs show focal recurrent imprinted domain hypermethylation. In this setting of shared physiologic erasure, NSEs harbor a malignancy-associated hypermethylation core, akin to that of a diverse cancer compendium. Beyond these concordances, we found subtype epigenetic homology with pluripotent versus differentiated states. ECs demonstrate a striking convergence of both CpG and CpH (non-CpG) methylation with pluripotent states; the pluripotential methyl-CpH signature crosses species boundaries and is distinct from neuronal methyl-CpH. EC differentiation to TE and YST entails reprogramming toward the somatic state, with loss of methyl-CpH but de novo methylation of pluripotency loci such as NANOG Extreme methyl-depletion among SE reflects the PGC methylation nadir. Adjacent to TGCTs, benign testis methylation profiles are determined by spermatogenetic proficiency measured by Johnsen score. In sum, TGCTs share collective entrapment in a PGC-like state of genomic-imprint and DPPA3 erasure, recurrent hypermethylation of cancer-associated targets, and subtype-dependent pluripotent, germline, or somatic methylation.

Human Germ Cell Tumors are Developmental Cancers: Impact of Epigenetics on Pathobiology and Clinic.

Current (high throughput omics-based) data support the model that human (malignant) germ cell tumors are not initiated by somatic mutations, but, instead through a defined locked epigenetic status, representative of their cell of origin. This elegantly explains the role of both genetic susceptibility as well as environmental factors in the pathogenesis, referred to as 'genvironment'. Moreover, it could also explain various epidemiological findings, including the rising incidence of this type of cancer in Western societies. In addition, it allows for identification of clinically relevant and informative biomarkers both for diagnosis and follow-up of individual patients. The current status of these findings will be discussed, including the use of high throughput DNA methylation profiling for determination of differentially methylated regions (DMRs) as well as chromosomal copy number variation (CNV). Finally, the potential value of methylation-specific tumor DNA fragments (i.e., XIST promotor) as well as embryonic microRNAs as molecular biomarkers for cancer detection in liquid biopsies will be presented.

Widespread somatic L1 retrotransposition occurs early during gastrointestinal cancer evolution.

Somatic L1 retrotransposition events have been shown to occur in epithelial cancers. Here, we attempted to determine how early somatic L1 insertions occurred during the development of gastrointestinal (GI) cancers. Using L1-targeted resequencing (L1-seq), we studied different stages of four colorectal cancers arising from colonic polyps, seven pancreatic carcinomas, as well as seven gastric cancers. Surprisingly, we found somatic L1 insertions not only in all cancer types and metastases but also in colonic adenomas, well-known cancer precursors. Some insertions were also present in low quantities in normal GI tissues, occasionally caught in the act of being clonally fixed in the adjacent tumors. Insertions in adenomas and cancers numbered in the hundreds, and many were present in multiple tumor sections, implying clonal distribution. Our results demonstrate that extensive somatic insertional mutagenesis occurs very early during the development of GI tumors, probably before dysplastic growth.

A pipeline to quantify serum and cerebrospinal fluid microRNAs for diagnosis and detection of relapse in paediatric malignant germ-cell tumours.

BACKGROUND: The current biomarkers alpha-fetoprotein and human chorionic gonadotropin have limited sensitivity and specificity for diagnosing malignant germ-cell tumours (GCTs). MicroRNAs (miRNAs) from the miR-371-373 and miR-302/367 clusters are overexpressed in all malignant GCTs, and some of these miRNAs show elevated serum levels at diagnosis. Here, we developed a robust technical pipeline to quantify these miRNAs in the serum and cerebrospinal fluid (CSF). The pipeline was used in samples from a cohort of exclusively paediatric patients with gonadal and extragonadal malignant GCTs, compared with appropriate tumour and non-tumour control groups. METHODS: We developed a method for miRNA quantification that enabled sample adequacy assessment and reliable data normalisation. We performed qRT-PCR profiling for miR-371-373 and miR-302/367 cluster miRNAs in a total of 45 serum and CSF samples, obtained from 25 paediatric patients. RESULTS: The exogenous non-human spike-in cel-miR-39-3p and the endogenous housekeeper miR-30b-5p were optimal for obtaining robust serum and CSF qRT-PCR quantification. A four-serum miRNA panel (miR-371a-3p, miR-372-3p, miR-373-3p and miR-367-3p): (i) showed high sensitivity/specificity for diagnosing paediatric extracranial malignant GCT; (ii) allowed early detection of relapse of a testicular mixed malignant GCT; and (iii) distinguished intracranial malignant GCT from intracranial non-GCT tumours at diagnosis, using CSF and serum samples. CONCLUSIONS: The pipeline we have developed is robust, scalable and transferable. It potentially promises to improve clinical management of paediatric (and adult) malignant GCTs.

Mutations in LRRC50 predispose zebrafish and humans to seminomas.

Seminoma is a subclass of human testicular germ cell tumors (TGCT), the most frequently observed cancer in young men with a rising incidence. Here we describe the identification of a novel gene predisposing specifically to seminoma formation in a vertebrate model organism. Zebrafish carrying a heterozygous nonsense mutation in Leucine-Rich Repeat Containing protein 50 (lrrc50 also called dnaaf1), associated previously with ciliary function, are found to be highly susceptible to the formation of seminomas. Genotyping of these zebrafish tumors shows loss of heterozygosity (LOH) of the wild-type lrrc50 allele in 44.4% of tumor samples, correlating with tumor progression. In humans we identified heterozygous germline LRRC50 mutations in two different pedigrees with a family history of seminomas, resulting in a nonsense Arg488* change and a missense Thr590Met change, which show reduced expression of the wild-type allele in seminomas. Zebrafish in vivo complementation studies indicate the Thr590Met to be a loss-of-function mutation. Moreover, we show that a pathogenic Gln307Glu change is significantly enriched in individuals with seminoma tumors (13% of our cohort). Together, our study introduces an animal model for seminoma and suggests LRRC50 to be a novel tumor suppressor implicated in human seminoma pathogenesis.

Endogenous Nodal signaling regulates germ cell potency during mammalian testis development.

Germ cells, the embryonic precursors of sperm or oocytes, respond to molecular cues that regulate their sex-specific development in the fetal gonads. In males in particular, the balance between continued proliferation and cell fate commitment is crucial: defects in proliferation result in insufficient spermatogonial stem cells for fertility, but escape from commitment and prolonged pluripotency can cause testicular germ cell tumors. However, the factors that regulate this balance remain unidentified. Here, we show that signaling by the TGFß morphogen Nodal and its co-receptor Cripto is active during a crucial window of male germ cell development. The Nodal pathway is triggered when somatic signals, including FGF9, induce testicular germ cells to upregulate Cripto. Germ cells of mutant mice with compromised Nodal signaling showed premature differentiation, reduced pluripotency marker expression and a reduced ability to form embryonic germ (EG) cell colonies in vitro. Conversely, human testicular tumors showed upregulation of NODAL and CRIPTO that was proportional to invasiveness and to the number of malignant cells. Thus, Nodal signaling provides a molecular control mechanism that regulates male germ cell potency in normal development and testicular cancer.

miMsg: a target enrichment algorithm for predicted miR-mRNA interactions based on relative ranking of matched expression data.

MOTIVATION: Algorithms predicting microRNA (miR)-mRNA interactions generate high numbers of possible interactions, many of which might be non-existent or irrelevant in a certain biological context. It is desirable to develop a transparent, user-friendly, unbiased tool to enrich miR-mRNA predictions. RESULTS: The miMsg algorithm uses matched miR/mRNA expression data to enrich miR-mRNA predictions. It grades interactions by the number, magnitude and significance of misplacements in the combined ranking profiles of miR/mRNA expression assessed over multiple biological samples. miMsg requires minimal user input and makes no statistical assumptions. It identified 921 out of 56 262 interactions as top scoring and significant in an actual germ cell cancer dataset. Twenty-eight miR-mRNA pairs were deemed of highest interest based on ranking by miMsg and supported by current knowledge about validated interactions and biological function. To conclude, miMsg is an effective algorithm to reduce a high number of predicted interactions to a small set of high confidence interactions for further study. AVAILABILITY AND IMPLEMENTATION: Matlab source code and datasets available at www.martinrijlaarsdam.nl/mimsg . SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Feeder-free culture of human pluripotent stem cells drives MDM4-mediated gain of chromosome 1q.

Culture-acquired variants in human pluripotent stem cells (hPSCs) hinder their applications in research and clinic. However, the mechanisms that underpin selection of variants remain unclear. Here, through analysis of comprehensive karyotyping datasets from over 23,000 hPSC cultures of more than 1,500 lines, we explored how culture conditions shape variant selection. Strikingly, we identified an association of chromosome 1q gains with feeder-free cultures and noted a rise in its prevalence in recent years, coinciding with increased usage of feeder-free regimens. Competition experiments of multiple isogenic lines with and without a chromosome 1q gain confirmed that 1q variants have an advantage in feeder-free (E8/vitronectin), but not feeder-based, culture. Mechanistically, we show that overexpression of MDM4, located on chromosome 1q, drives variants' advantage in E8/vitronectin by alleviating genome damage-induced apoptosis, which is lower in feeder-based conditions. Our study explains condition-dependent patterns of hPSC aberrations and offers insights into the mechanisms of variant selection.

Analysis of a mouse germ cell tumor model establishes pluripotency-associated miRNAs as conserved serum biomarkers for germ cell cancer detection.

Malignant testicular germ cells tumors (TGCTs) are the most common solid cancers in young men. Current TGCT diagnostics include conventional serum protein markers, but these lack the sensitivity and specificity to serve as accurate markers across all TGCT subtypes. MicroRNAs (miRNAs) are small non-coding regulatory RNAs and informative biomarkers for several diseases. In humans, miRNAs of the miR-371-373 cluster are detectable in the serum of patients with malignant TGCTs and outperform existing serum protein markers for both initial diagnosis and subsequent disease monitoring. We previously developed a genetically engineered mouse model featuring malignant mixed TGCTs consisting of pluripotent embryonal carcinoma (EC) and differentiated teratoma that, like the corresponding human malignancies, originate in utero and are highly chemosensitive. Here, we report that miRNAs in the mouse miR-290-295 cluster, homologs of the human miR-371-373 cluster, were detectable in serum from mice with malignant TGCTs but not from tumor-free control mice or mice with benign teratomas. miR-291-293 were expressed and secreted specifically by pluripotent EC cells, and expression was lost following differentiation induced by the drug thioridazine. Notably, miR-291-293 levels were significantly higher in the serum of pregnant dams carrying tumor-bearing fetuses compared to that of control dams. These findings reveal that expression of the miR-290-295 and miR-371-373 clusters in mice and humans, respectively, is a conserved feature of malignant TGCTs, further validating the mouse model as representative of the human disease. These data also highlight the potential of serum miR-371-373 assays to improve patient outcomes through early TGCT detection, possibly even prenatally.

Integrated genomic analysis reveals aberrations in WNT signaling in germ cell tumors of childhood and adolescence.

Germ cell tumors (GCTs) are neoplasms of the testis, ovary and extragonadal sites that occur in infants, children, adolescents and adults. Post-pubertal (type II) malignant GCTs may present as seminoma, non-seminoma or mixed histologies. In contrast, pre-pubertal (type I) GCTs are limited to (benign) teratoma and (malignant) yolk sac tumor (YST). Epidemiologic and molecular data have shown that pre- and post-pubertal GCTs arise by distinct mechanisms. Dedicated studies of the genomic landscape of type I and II GCT in children and adolescents are lacking. Here we present an integrated genomic analysis of extracranial GCTs across the age spectrum from 0-24 years. Activation of the WNT pathway by somatic mutation, copy-number alteration, and differential promoter methylation is a prominent feature of GCTs in children, adolescents and young adults, and is associated with poor clinical outcomes. Significantly, we find that small molecule WNT inhibitors can suppress GCT cells both in vitro and in vivo. These results highlight the importance of WNT pathway signaling in GCTs across all ages and provide a foundation for future efforts to develop targeted therapies for these cancers.

Chromosome 3p25.3 Gain Is Associated With Cisplatin Resistance and Is an Independent Predictor of Poor Outcome in Male Malignant Germ Cell Tumors.

PURPOSE: Cisplatin is the main systemic treatment modality for male type II germ cell tumors (GCTs). Although generally very effective, 5%-10% of patients suffer from cisplatin-resistant disease. Identification of the driving mechanisms of resistance will enable improved risk stratification and development of alternative treatments. METHODS: We developed and characterized cisplatin-resistant GCT cell line models and compared their molecular characteristics with patient samples with cisplatin resistance and/or a poor clinical outcome. Subsequently, the association between the overlapping genetic features and clinical data was assessed. Finally, we used Cox regression to determine the prognostic relevance of these features within the currently used risk classification. RESULTS: Gain of chromosome 3p25.3 was detected in all cisplatin-resistant cell lines, and copy number of this region correlated with the level of resistance (R = 0.96, P = 1.5e-04). Gain of this region was detected at low frequencies in primary tumors and at higher frequencies in relapsed and/or cisplatin-resistant tumors. Chromosome 3p25.3 gain was associated with shorter progression-free survival and overall survival, with the strongest association observed in nonseminomas excluding pure teratomas. 3p25.3 gain was more frequently observed in tumors with yolk sac tumor histology and predicted adverse outcome independent of the International Germ Cell Cancer Collaborative Group risk classification and the presence of TP53/MDM2 alterations. CONCLUSION: On the basis of both in vitro analyses and clinical data, we found 3p25.3 to be strongly associated with cisplatin resistance and poor clinical outcome in male type II GCTs. Using genomic profiling, 3p25.3 status could help to improve risk stratification in male patients with type II GCT. Further characterization of this locus and underlying mechanisms of resistance is warranted to guide development of novel treatment approaches for cisplatin-resistant disease.

Diagnosis of testicular carcinoma in situ '(intratubular and microinvasive)' seminoma and embryonal carcinoma using direct enzymatic alkaline phosphatase reactivity on frozen histological sections.

AIMS: Testis-sparing surgery might benefit quality of life, but can only be applied with histological examination for the presence of invasive germ cell tumour components, and the precursor carcinoma in situ (CIS). Currently, diagnosis is based on paraffin-embedded tissue, therefore a delay in further surgery is mainly unavoidable. The aim was to develop an intraoperative assessment technique using alkaline phosphatase as a marker. METHODS AND RESULTS: A total of 4093 snap-frozen samples and matched paraffin-embedded tissue of 1500 patients were included. Besides standard haematoxylin and eosin (H&E) staining, the direct enzymatic alkaline phosphatase reactivity (dAP) test (duration 15 min) was applied on frozen sections, while H&E and immunohistochemistry for detection of OCT3/4, α-fetoprotein, human chorionic gonadotrophin (hCG) and cytokeratin was performed on the paraffin-embedded slides. Endothelial cells served as control for the dAP test. Positive staining was found in all CIS (n=965), seminoma (n=1035) and embryonal carcinoma (n=584), either intratubular, microinvasive or invasive. Differentiated non-seminomas (n=1238) showed variable staining. No staining was identified in spermatocytic seminomas (n=5), testicular lymphomas (n=42), testicular rhabdomyosarcomas (n=7), Leydig cell tumours (n=31), Sertoli-cell-only nodules (n=4), (epi) dermoid cyst (n=16), normal testicular parenchyma (n=116), testicular torsion (n=32) and inflammation of the epididymis (n=19). The dAP test results matched H&E-stained parallel sections, as well paraffin-embedded tissue analysis, including immunohistochemistry. CONCLUSIONS: The dAP test is an informative, reproducible and easy tool to diagnose CIS, (intratubular and microinvasive) seminoma and embryonal carcinoma on frozen tissue sections, being of great value in the context of sparing surgery.

Histopathological and molecular features of late relapses in non-seminomas.

OBJECTIVE: • To describe the histopathological types of late relapses of germ cell tumours and to search for molecular markers associated with chemotherapy resistance. PATIENTS AND METHODS: • Samples from 14 patients with late relapse from a non-seminoma were analysed. • Archival tumour tissue was gathered at intial diagnosis (n= 9) and at relapse (n= 9), mostly after previous treatment with chemotherapy. • In addition to routine histopathology, tumours were analysed for microsatellite instability and screened for mutations in the KRAS and BRAF genes. RESULTS: • Relapse occurred after 76.5 months (median, range: 24-209 months). • The histology in relapse was pure yolk sac tumour in four of the nine patients analysed. • Three had a non-germ cell malignancy, one was a mixed non-seminoma and one was a pure mature teratoma. • One sample with non-germ cell malignancy originated from a yolk sac tumour without any evidence of teratoma. • In four of 12 evaluable patients, high-level microsatellite instability was observed. • All patients were KRAS wild-type but four showed a BRAF mutation at V600E. CONCLUSIONS: • Many late relapses of germ cell tumours show pure yolk sac histology. • Non-germ cell malignancies do not necessarily develop from teratoma but can also arise from yolk sac histology. • The biology underlying chemotherapy resistance in late relapse could be related to a high incidence of microsatellite instability and BRAF mutation V600E, which were found in half of the patients.

Global DNA methylation in fetal human germ cells and germ cell tumours: association with differentiation and cisplatin resistance.

Differences in the global methylation pattern, ie hyper- as well as hypo-methylation, are observed in cancers including germ cell tumours (GCTs). Related to their precursor cells, GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n = 103) and GCTs (n = 251) by immunohistochemical staining for 5-(m)cytidine. The global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, whereas female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, intratubular germ cell neoplasia unclassified, and gonadoblastomas) are hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumours, and choriocarcinomas) show a higher degree of methylation. Embryonal carcinomas show an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam-2 before and after demethylation using 5-azacytidine. Exposure to 5-azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell-specific marker VASA, showed increased expression. Following treatment with 5-azacytidine, TCam-2 cells were analysed using a high-throughput methylation screen for changes in the methylation sites of 14,000 genes. Among the genes revealing changes, interesting targets were identified: ie demethylation of KLF11, a putative tumour suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs.