Dominik Schneider

Genomic and expression profiling of human spermatocytic seminomas: primary spermatocyte as tumorigenic precursor and DMRT1 as candidate chromosome 9 gene.

Spermatocytic seminomas are solid tumors found solely in the testis of predominantly elderly individuals. We investigated these tumors using a genome-wide analysis for structural and numerical chromosomal changes through conventional karyotyping, spectral karyotyping, and array comparative genomic hybridization using a 32 K genomic tiling-path resolution BAC platform (confirmed by in situ hybridization). Our panel of five spermatocytic seminomas showed a specific pattern of chromosomal imbalances, mainly numerical in nature (range, 3-24 per tumor). Gain of chromosome 9 was the only consistent anomaly, which in one case also involved amplification of the 9p21.3-pter region. Parallel chromosome level expression profiling as well as microarray expression analyses (Affymetrix U133 plus 2.0) was also done. Unsupervised cluster analysis showed that a profile containing transcriptional data on 373 genes (difference of > or = 3.0-fold) is suitable for distinguishing these tumors from seminomas/dysgerminomas. The diagnostic markers SSX2-4 and POU5F1 (OCT3/OCT4), previously identified by us, were among the top discriminatory genes, thereby validating the experimental set-up. In addition, novel discriminatory markers suitable for diagnostic purposes were identified, including Deleted in Azospermia (DAZ). Although the seminomas/dysgerminomas were characterized by expression of stem cell-specific genes (e.g., POU5F1, PROM1/CD133, and ZFP42), spermatocytic seminomas expressed multiple cancer testis antigens, including TSP50 and CTCFL (BORIS), as well as genes known to be expressed specifically during prophase meiosis I (TCFL5, CLGN, and LDHc). This is consistent with different cells of origin, the primordial germ cell and primary spermatocyte, respectively. Based on the region of amplification defined on 9p and the associated expression plus confirmatory immunohistochemistry, DMRT1 (a male-specific transcriptional regulator) was identified as a likely candidate gene for involvement in the development of spermatocytic seminomas.

An Immunodeficiency Disease with RAG Mutations and Granulomas

We describe three unrelated girls who had an immunodeficiency disease with granulomas in the skin, mucous membranes, and internal organs. All three girls had severe complications after viral infections, including B-cell lymphoma associated with Epstein-Barr virus (EBV). Other findings were hypogammaglobulinemia, a diminished number of T and B cells, and sparse thymic tissue on ultrasonography. Molecular analysis revealed that the patients were compound heterozygotes for mutations in recombination activating gene 1 or 2 (RAG1 or RAG2). In each case, both parents were heterozygous carriers of a RAG mutation. The mutations were associated with reduced function of RAG in vitro (3 to 30% of normal activity). The parents and one sibling in the three families were healthy.

Further characterization of the first seminoma cell line TCam-2

Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam‐2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q‐PCR) and NANOG, and the absence of CD30, SSX2‐4, and SOX2, confirms that TCam‐2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam‐2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam‐2 is the first well‐characterized seminoma‐derived cell line, with an exceptional mutation, rarely found in TGCTs.

Phylogenetic Analysis of a Microbialite-Forming Microbial Mat from a Hypersaline Lake of the Kiritimati Atoll, Central Pacific

On the Kiritimati atoll, several lakes exhibit microbial mat-formation under different hydrochemical conditions. Some of these lakes trigger microbialite formation such as Lake 21, which is an evaporitic, hypersaline lake (salinity of approximately 170‰). Lake 21 is completely covered with a thick multilayered microbial mat. This mat is associated with the formation of decimeter-thick highly porous microbialites, which are composed of aragonite and gypsum crystals. We assessed the bacterial and archaeal community composition and its alteration along the vertical stratification by large-scale analysis of 16S rRNA gene sequences of the nine different mat layers. The surface layers are dominated by aerobic, phototrophic, and halotolerant microbes. The bacterial community of these layers harbored Cyanobacteria (Halothece cluster), which were accompanied with known phototrophic members of the Bacteroidetes and Alphaproteobacteria. In deeper anaerobic layers more diverse communities than in the upper layers were present. The deeper layers were dominated by Spirochaetes, sulfate-reducing bacteria (Deltaproteobacteria), Chloroflexi (Anaerolineae and Caldilineae), purple non-sulfur bacteria (Alphaproteobacteria), purple sulfur bacteria (Chromatiales), anaerobic Bacteroidetes (Marinilabiacae), Nitrospirae (OPB95), Planctomycetes and several candidate divisions. The archaeal community, including numerous uncultured taxonomic lineages, generally changed from Euryarchaeota (mainly Halobacteria and Thermoplasmata) to uncultured members of the Thaumarchaeota (mainly Marine Benthic Group B) with increasing depth.

Land-use choices follow profitability at the expense of ecological functions in Indonesian smallholder landscapes

Smallholder-dominated agricultural mosaic landscapes are highlighted as model production systems that deliver both economic and ecological goods in tropical agricultural landscapes, but trade-offs underlying current land-use dynamics are poorly known. Here, using the most comprehensive quantification of land-use change and associated bundles of ecosystem functions, services and economic benefits to date, we show that Indonesian smallholders predominantly choose farm portfolios with high economic productivity but low ecological value. The more profitable oil palm and rubber monocultures replace forests and agroforests critical for maintaining above- and below-ground ecological functions and the diversity of most taxa. Between the monocultures, the higher economic performance of oil palm over rubber comes with the reliance on fertilizer inputs and with increased nutrient leaching losses. Strategies to achieve an ecological-economic balance and a sustainable management of tropical smallholder landscapes must be prioritized to avoid further environmental degradation.

Regional deep hyperthermia for salvage treatment of children and adolescents with refractory or recurrent non-testicular malignant germ-cell tumours: an open-label, non-randomised, single-institution, phase 2 study

BACKGROUND Although the survival of children and adolescents with malignant germ-cell tumours has improved greatly in recent years, the outcome remains poor for those with refractory or recurrent malignant germ-cell tumours. We aimed to determine whether objective tumour response could be achieved in patients with refractory or recurrent malignant germ-cell tumours with PEI-regional deep hyperthermia as salvage treatment. METHODS Patients with refractory or recurrent non-testicular malignant germ-cell tumours after standard cisplatin-based chemotherapy were treated prospectively with PEI chemotherapy (cisplatin 40 mg/m(2), delivered intravenously on days 1 and 4; etoposide 100 mg/m(2), intravenously on days 1-4; and ifosfamide 1800 mg/m(2), intravenously on days 1-4) plus simultaneous 1-h regional deep hyperthermia (41-43°C) on days 1 and 4. Patients received three to four treatment courses at 21-day intervals until residual tumour resection was possible; they subsequently received one or two additional courses of PEI-regional deep hyperthermia. Local radiotherapy was given for incompletely resected tumours. Chemotherapy and hyperthermia toxic effects were assessed using WHO grading. The primary endpoint was the proportion of patients who had an objective response as assessed with Response Evaluation Criteria in Solid Tumors version 1.0 guidelines. Secondary endpoints were the event-free survival and overall survival after 5 years. This ongoing PEI-regional deep hyperthermia study (Hyper-PEI protocol) is registered at the German Cancer Society, number 50-2732. FINDINGS 44 patients aged 7 months to 21 years (median 2 years 7 months) with refractory or recurrent malignant germ-cell tumours (nine patients with poor response, 23 patients with first relapse, 12 patients with multiple relapses) were included in this study. We identified 34 yolk sac tumours, eight embryonal carcinomas, one choriocarcinoma, and one dysgerminoma by histology analysis. Of the 35 patients who had sufficient clinical and radiographical data available for response assessment, 30 (86%) had an objective response to treatment (16 patients had complete remission and 14 had partial remission). 5-year event-free survival was 62% (95% CI 45-75), and 5-year overall survival was 72% (95% CI 55-83). The median follow-up of surviving patients was 82 months (range 9-195). WHO grade 3-4 neutropenia and thrombocytopenia occurred in all 181 chemotherapy cycles. Granulocytopenic fever, which required intercurrent hospital admission, was noted in 29 (66%) of 44 patients after 53 (29%) of 181 courses. Five patients experienced treatment-related grade-3 acute renal toxic effects. INTERPRETATION A multimodal strategy integrating PEI-regional deep hyperthermia and tumour resection with or without radiation can successfully treat children and adolescents with refractory or recurrent malignant non-testicular germ-cell tumours. The long-term prognosis of patients with poor response or after first relapse was almost similar to those receiving first-line treatment. This strategy merits further investigation. FUNDING Deutsche Krebshilfe eV, Bonn, Elterninitiative Kinderkrebsklinik Düsseldorf eV, the Barbara and Hubertus-Trettnerstiftung, and the Marie Quendt Fund.

Rare cancers in children - The EXPeRT Initiative: a report from the European Cooperative Study Group on Pediatric Rare Tumors.

The low incidence and the heterogeneity of very rare tumors (VRTs) demand for international cooperation. In 2008, EXPeRT (European Cooperative Study Group for Pediatric Rare Tumors) was founded by national groups from Italy, France, United Kingdom, Poland and Germany. The first aims of EXPeRT were to agree on a uniform definition of VRTs and to develop the currently most relevant scientific questions. Current initiatives include international data exchange, retrospective and prospective studies of specific entities, and the development of harmonized and internationally recognized guidelines. Moreover, EXPeRT established a network for expert consultation to assist in clinical decision in VRTs.

Prevalence of c-KIT Mutations in Gonadoblastoma and Dysgerminomas of Patients with Disorders of Sex Development (DSD) and Ovarian Dysgerminomas

Activating c-KIT mutations (exons 11 and 17) are found in 10–40% of testicular seminomas, the majority being missense point mutations (codon 816). Malignant ovarian dysgerminomas represent ∼3% of all ovarian cancers in Western countries, resembling testicular seminomas, regarding chromosomal aberrations and c-KIT mutations. DSD patients with specific Y-sequences have an increased risk for Type II Germ Cell Tumor/Cancer, with gonadoblastoma as precursor progressing to dysgerminoma. Here we present analysis of c-KIT exon 8, 9, 11, 13 and 17, and PDGFRA exon 12, 14 and 18 by conventional sequencing together with mutational analysis of c-KIT codon 816 by a sensitive and specific LightCycler melting curve analysis, confirmed by sequencing. The results are combined with data on TSPY and OCT3/4 expression in a series of 16 DSD patients presenting with gonadoblastoma and dysgerminoma and 15 patients presenting pure ovarian dysgerminomas without DSD. c-KIT codon 816 mutations were detected in five out of the total of 31 cases (all found in pure ovarian dysgerminomas). A synonymous SNP (rs 5578615) was detected in two patients, one DSD patient (with bilateral disease) and one patient with dysgerminoma. Next to these, three codon N822K mutations were detected in the group of 15 pure ovarian dysgerminomas. In total activating c-KIT mutations were found in 53% of ovarian dysgerminomas without DSD. In the group of 16 DSD cases a N505I and D820E mutation was found in a single tumor of a patient with gonadoblastoma and dysgerminoma. No PDGFRA mutations were found. Positive OCT3/4 staining was present in all gonadoblastomas and dysgerminomas investigated, TSPY expression was only seen in the gonadoblastoma/dysgerminoma lesions of the 16 DSD patients. This data supports the existence of two distinct but parallel pathways in the development of dysgerminoma, in which mutational status of c-KIT might parallel the presence of TSPY.

Impact of Lowland Rainforest Transformation on Diversity and Composition of Soil Prokaryotic Communities in Sumatra (Indonesia).

Prokaryotes are the most abundant and diverse group of microorganisms in soil and mediate virtually all biogeochemical cycles in terrestrial ecosystems. Thereby, they influence aboveground plant productivity and diversity. In this study, the impact of rainforest transformation to intensively managed cash crop systems on soil prokaryotic communities was investigated. The studied managed land use systems comprised rubber agroforests (jungle rubber), rubber plantations and oil palm plantations within two Indonesian landscapes Bukit Duabelas and Harapan. Soil prokaryotic community composition and diversity were assessed by pyrotag sequencing of bacterial and archaeal 16S rRNA genes. The curated dataset contained 16,413 bacterial and 1679 archaeal operational taxonomic units at species level (97% genetic identity). Analysis revealed changes in indigenous taxon-specific patterns of soil prokaryotic communities accompanying lowland rainforest transformation to jungle rubber, and intensively managed rubber and oil palm plantations. Distinct clustering of the rainforest soil communities indicated that these are different from the communities in the studied managed land use systems. The predominant bacterial taxa in all investigated soils were Acidobacteria, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Overall, the bacterial community shifted from proteobacterial groups in rainforest soils to Acidobacteria in managed soils. The archaeal soil communities were mainly represented by Thaumarchaeota and Euryarchaeota. Members of the Terrestrial Group and South African Gold Mine Group 1 (Thaumarchaeota) dominated in the rainforest and members of Thermoplasmata in the managed land use systems. The alpha and beta diversity of the soil prokaryotic communities was higher in managed land use systems than in rainforest. In the case of bacteria, this was related to soil characteristics such as pH value, exchangeable Ca and Fe content, C to N ratio, and extractable P content. Archaeal community composition and diversity were correlated to pH value, exchangeable Fe content, water content, and total N. The distribution of bacterial and archaeal taxa involved in biological N cycle indicated functional shifts of the cycle during conversion of rainforest to plantations.

Ovarian Sertoli Leydig cell tumours in children and adolescents: An analysis of the European Cooperative Study Group on Pediatric Rare Tumors (EXPeRT)

OBJECTIVE To analyse ovarian Sertoli-Leydig cell tumours (SLCTs) for potential prognostic markers and their use for treatment stratification. PATIENTS Forty-four patients were included. Patients were prospectively reported to the German MAKEI (Maligne Keimzelltumoren) studies (n=23), French TGM protocols (n=10), Italian Rare Tumour Project (TREP) registry (n=6), and the Polish Pediatric Rare Tumour Study group (n=5). Tumours were classified according to World Health Organisation (WHO) and staged according to International Federation of Gynecological Oncology (FIGO). RESULTS Median age was 13.9 (0.5-17.4) years. All patients underwent resection by tumour enucleation (n=8), ovariectomy (n=17), adenectomy isolated (n=18) or with hysterectomy (n=1). FIGO-stage: Ia 24pts., Ic 17pts., II/III 3pts. One patient had bilateral tumours. Four patients (stage Ia: 3, stage Ic: 1) developed a metachronous contralateral tumour. Otherwise, all stage Ia patients remained in complete remission. Among 20 patients with incomplete resection or tumour spread (stage Ic-III), eight relapsed, and five patients died. Eleven patients were initially treated with two to sixcycles of cisplatin-based chemotherapy. Of these, seven patients are in continuous remission. Poor histological differentiation was associated with higher relapse rate (5/13) compared to intermediate (3/18) and high differentiation (0/4). Tumours with retiform pattern or heterologous elements showed a high relapse rate, too (5/11). After a median follow-up of 62 months, event-free survival is 0.70±0.07, relapse-free survival 0.81±0.06 and overall survival 0.87±0.05. CONCLUSIONS Prognosis of SLCTs is determined by stage and histopathologic differentiation. Complete resection with careful avoidance of spillage is a prerequisite of cure. The impact of chemotherapy in incompletely resected and advanced stage tumours remains to be evaluated.