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INTRODUCTION: The management of complex cysts of the breast is an ongoing topic of discussion. The aim of this study was to determine the prevalence of underlying malignancy in radiologically diagnosed complex cysts, and to assess whether watchful waiting could be the preferred method to safely manage complex cysts of the breast. SUBJECTS AND METHODS: A single-center retrospective study was performed between May 2003 and November 2019 in the VieCuri Medical Centre. Women with a radiologically diagnosed complex cyst of the breast were included. Prevalence of underlying malignancy was calculated, as were absolute risk reduction and number needed to treat in order to diagnose malignancy. In addition, patient characteristics were compared to determine characteristics associated with malignancy. RESULTS: Of 78 radiologically diagnosed complex cysts of the breast, five (6,4%) were found to be malignant. The number needed to treat was calculated at 12,8 (absolute riks reduction 0,078). Age (P = 0,003) was associated with malignancy. CONCLUSION: Complex cysts of the breast could be managed more conservatively. Patient characteristics can be used to assess the eligibility for radiological follow-up. This, in turn, would lead to a lower NNT and possibly a decrease in disease burden and healthcare costs.

Mycoplasma pneumoniae and Streptococcus pneumoniae are the most common bacterial causes of pneumonia in children. The clinical characteristics of pneumonia differ significantly between the two bacteria. We aimed to elucidate the differences in pathogenesis between M. pneumoniae and S. pneumoniae by characterizing the respiratory epithelial cell immune response to both pathogens. Using primary human bronchial epithelial cells in air-liquid interface cultures, we observed lower production of the proinflammatory cytokines interleukin-6 (IL-6) and IL-8 in response to M. pneumoniae than to S. pneumoniae. In contrast to the differences in proinflammatory cytokine production, Toll-like receptor 2 (TLR2)-mediated signaling in response to M. pneumoniae was stronger than to S. pneumoniae. This difference largely depended on TLR1 and not TLR6. We found that M. pneumoniae, but not S. pneumoniae, also induced signaling of TLR10, a coreceptor of TLR2 that has inhibitory properties. M. pneumoniae-induced TLR10 signaling on airway epithelial cells was partially responsible for low IL-8 production, as blocking TLR10 by specific antibodies increased cytokine production. M. pneumoniae maintained Th2-associated cytokine production by epithelial cells, which concurs with the known association of M. pneumoniae infection with asthma. M. pneumoniae left IL-33 levels unchanged, whereas S. pneumoniae downregulated IL-33 production both under homeostatic and Th2-promoting conditions. By directly comparing M. pneumoniae and S. pneumoniae, we demonstrate that M. pneumoniae avoids induction of proinflammatory cytokine response despite its ability to induce robust TLR2 signaling. Our new findings suggest that this apparent paradox may be partially explained by M. pneumoniae-induced signaling of TLR2/TLR10.

Present field experiment was conducted at farmer’s field in Ringondiya village, Madhya Pradesh during rabi season 2018-19 to study the effect of integrated nutrient management practices on performance of chickpea, basic soil properties and nutrient availability. The nutrient concentration, uptake and protein content of chickpea (cv. JG-322) was evaluated under seven treatments viz., T1-Control, T2-100% N:P:K (20:50:20), T3-50% N:P:K + FYM @5 t ha-1, T4-50% N:P:K + vermicompost @2 t ha-1, T5-50% N:P:K + PSB @4 kg ha-1, T6-50% N:P:K + FYM @5 t ha-1 + PSB @4 kg ha-1 and T7-50% N:P:K + vermicompost @2 t ha-1 +PSB @4 kg ha-1 replicated thrice in a randomized block design. The N, P, K and S concentration in seed and straw, nutrient removal by seed, straw and total and protein content at harvest stage were determined. The results revealed that the integrated nutrient management practice significantly improved the nutrient concentration, uptake and quality of chickpea.

Present field experiment was conducted at farmer’s field in Ringondiya village, Madhya Pradesh during rabi season 2018-19 to study the effect of integrated nutrient management practices on performance of chickpea, basic soil properties and nutrient availability. The performance of chickpea (cv. JG-322) was evaluated under seven treatments viz., T1-Control, T2-100% N:P:K (20:50:20), T3-50% N:P:K + FYM @5 t ha-1, T4-50% N:P:K + vermicomposting @2 t ha-1, T5-50% N:P:K + PSB @4 kg ha-1, T6-50% N:P:K + FYM @5 t ha-1 + PSB @4 kg ha-1 and T7-50% N:P:K + vermicomposting @2 t ha-1 +PSB @4 kg ha-1 replicated thrice in a randomized block design. The grain yield, straw yield and harvest index of chickpea were determined at harvest. Similarly, the soil pH, electrical conductivity, soil organic carbon and soil available nutrients (N, P and K) were also determined in post harvest soil samples. The results revealed that the integrated nutrient management practice significantly improved the performance of chickpea. The soil organic carbon and available nutrients were also found increased under INM practices.

Over the years, the demand for ART with donated embryos has increased. Treatment can be performed using donated ‘surplus embryos’ from IVF treatment or with embryos intentionally created through so-called ‘double gamete donation’. Embryo donation is particularly sensitive because treatment results in the absence of a genetic link between the parent(s) and the child, creating complex family structures, including full genetic siblings living in another family in the case of surplus embryo donation. In this paper, we explore the ethical acceptability of embryo donation in light of the similarities and differences between surplus embryo donation and double gamete donation. We will argue that no overriding objections to either form of embryo donation exist. First of all, ART with donated embryos respects patients’ reproductive autonomy by allowing them to experience gestational parenthood. It also respects IVF patients’ reproductive autonomy by providing an additional option to discarding or donating surplus embryos to research. Second, an extensive body of empirical research has shown that a genetic link between parent and child is not a condition for a loving caring relationship between parent(s) and child. Third, the low moral status of a pre-implantation embryo signifies no moral duty for clinics to first use available surplus embryos or to prevent the development of (more) surplus embryos through double gamete donation. Fourth, there is no reason to assume that knowledge of having (full or half) genetically related persons living elsewhere provides an unacceptable impact on the welfare of donor-conceived offspring, existing children of the donors, and their respective families. Thus, patients and clinicians should discuss which form of ART would be suitable in their specific situation. To guarantee ethically sound ART with donated embryos certain conditions have to be met. Counselling of IVF patients should involve a discussion on the destination of potential surplus embryos. When counselling donors and recipient(s) a discussion of the significance of early disclosure of the child’s mode of conception, the implications of having children raised in families with whom they share no genetic ties, expectations around information-exchange and contact between donor and recipient families or genetically related siblings is warranted. Importantly, conclusions are mainly drawn from results of empirical studies on single gamete donation families. To evaluate the welfare of families created through surplus embryo donation or double gamete donation additional empirical research on these particular families is warranted.

The 5th edition of the World Health Organization Classification of CNS tumors defines the CNS neuroblastomaOn the basis of a previously published cohort of tumors molecularly classified as CNS neuroblastoma-Twenty-five patients were evaluated (13 girls; median age, 4.5 years). The tumors were often large (mean, 115 [ SD, 83] mL), showed no (24%) or limited (60%) perilesional edema, demonstrated heterogeneous enhancement, were often calcified and/or hemorrhagic (52%), were always T2WI-hyperintense to GM, and commonly had cystic and/or necrotic components (96%). The mean ADC values were low (687.8 [SD 136.3] × 10In our cohort, CNS neuroblastoma

BACKGROUND AND PURPOSE: The 5th edition of the World Health Organization Classification of CNS tumors defines the CNS neuroblastoma FOXR2 in the group of embryonal tumors. Published clinical outcomes tend to suggest a favorable outcome after resection, craniospinal irradiation, and chemotherapy. This multicenter study aimed to describe imaging features of CNS neuroblastoma-FOXR2, which have been poorly characterized thus far.MATERIALS AND METHODS: On the basis of a previously published cohort of tumors molecularly classified as CNS neuroblastoma-FOXR2, patients with available imaging data were identified. The imaging features on preoperative MR imaging and CT data were recorded by 8 experienced pediatric neuroradiologists in consensus review meetings.RESULTS: Twenty-five patients were evaluated (13 girls; median age, 4.5 years). The tumors were often large (mean, 115 [ SD, 83] mL), showed no (24%) or limited (60%) perilesional edema, demonstrated heterogeneous enhancement, were often calcified and/or hemorrhagic (52%), were always T2WI-hyperintense to GM, and commonly had cystic and/or necrotic components (96%). The mean ADC values were low (687.8 [SD 136.3] × 10-6 mm2/s). The tumors were always supratentorial. Metastases were infrequent (20%) and, when present, were of nodular appearance and leptomeningeal.CONCLUSIONS: In our cohort, CNS neuroblastoma FOXR2 tumors showed imaging features suggesting high-grade malignancy and, at the same time, showed characteristics of less aggressive behavior. There are important differential diagnoses, but the results of this study may assist in considering this diagnosis preoperatively.

Background Ependymoma is the third most common brain tumor in children. At the moment, surgery and radiotherapy are the only effective treatments that can be offered, and despite this, a significant part of the patients relapse with no therapeutic salvage options. Therefore, new treatment modalities are needed. To develop immunotherapies for these children, knowledge of the tumor microenvironment is crucial. The current study aims to unravel the tumor immune microenvironment (TIME) of pediatric posterior fossa A (PFA) ependymomas. Materials and Methods We used bulk RNA sequencing data of 22 pediatric ependymomas. We defined two groups, hereafter called PFA immune+ (PFAI+) and PFAI-, based on the RNA expression levels of the NanoString panel of Human PanCancer Immune Profiling genes. We performed gene set enrichment analysis and deconvoluted the bulk RNA samples with ependymoma-specific single-cell RNA sequencing datasets. To validate our findings on a protein level, we applied immunohistochemistry with antibodies recognizing tumor-infiltrating lymphocytes, tumor-associated macrophages and microglia. Results Unsupervised hierarchical clustering of RNA expression of immune-related genes revealed two distinct PFA groups. Differential gene expression analysis showed that PFAI+ have a significantly higher expression of genes associated with immune functions, such as CD3E, CCR2, GZMA, CXCL9 and TRBC2. Accordingly, gene set enrichment analysis demonstrated that several immune pathways, including T-cell signalling, interferon-gamma response and TNFα signalling are enriched in PFAI+ ependymomas. RNA expression of immune checkpoints was also higher in PFAI+ tumors, indicating that these tumors might be more responsive to combinational therapies including immune checkpoint inhibitors. While immunohistochemistry showed low amounts of infiltrating CD3+, CD8+ and CD20+ cells, high numbers of CD163+ and HLA-DRA+ cells were detected. These cells were mainly located in regions of tumor necrosis. Increased amounts of CD4+ and CD8+ lymphocytes were present in PFAI+ tumors compared to PFAI- tumors. Deconvolution of the bulk RNA samples based on single-cell RNA sequencing data revealed an enrichment of myeloid cell populations, especially microglia and macrophages. Furthermore, PFAI+ tumors were found to contain significantly higher relative proportions of T-cells compared to PFAI- tumors (median of 3.76% for PFAI+ compared to 0.03% for PFAI-). Conclusions We suggest that pediatric posterior fossa A ependymomas can be divided into two groups based on the expression of immune-related genes, in which PFAI+ ependymomas are characterized by higher RNA expression levels of these genes and greater amounts of tumor-infiltrating immune cells. Several techniques showed an enrichment of T-lymphocytes in the PFAI+ ependymomas relative to the PFAI- ependymomas. Disclosure Information J. Lammers: None. F. Calkoen: None. M. Kranendonk: None. A. Federico: None. M. Kool: None. L. Kester: None. J. van der Lugt: None.

The mortality and morbidity of patients with congenital diaphragmatic hernia (CDH) is primarily caused by treatment-resistant, persistent pulmonary hypertension. Structural vascular changes, exemplified by extensive muscularization, are already present early in gestation, but the origin of these abnormalities is unknown. Understanding the origin of the vascular defects is important to improve treatment modalities. Here, we show that the distribution of pericytes is different and may thereby potentially initiate the vascular pathology in CDH. Transient inhibition of retinoic acid (RA) signaling early during pregnancy, the basis of the CDH mouse model, led to an increase in the number of pericytes, thereby affecting the angiogenic potential of pericytes in the fetuses. Pericytes of CDH lungs showed reduced proliferation and an increased ACTA2 expression, which indicates that these pericytes are more contractile than in control lung pericytes. This resulted in increased pericyte coverage of pulmonary vessels and reduced expansion of the capillary bed, the earliest pathological sign of the structural changes in CDH. Furthermore, the pericytes had reduced and altered collagen IV deposition in CDH, pointing to a loss of basal membrane integrity between pericytes and endothelial cells. Inhibition of RA signaling in vitro resulted in reduced migration of pericytes, reduced angiogenesis, and loss of collagen IV expression. Importantly, we confirmed our findings in lungs of human CDH patient samples. In summary, inhibition of RA signaling affects the lung pericyte population, leading to increased contractility, reduced pulmonary angiogenesis, and aberrant lung development, as observed in CDH.

Additional file 1: Figure 1. Effects of CXCL9 and IL-8 plasma levels on patient survival. Figure 2. Plasma concentrations of IL-1β and IL-6 Inflammatory markers in plasma of PAH and CTEPH patients at diagnosis and 1-year follow up. Figure 3. Inflammatory marker concentration of PH patients at diagnosis and at 1-year follow up are not separated by principal component analysis. Figure 4. Contribution of inflammatory markers and clinical parameters to multivariate analyses. Figure 5. Combination of inflammatory markers and clinical parameters leads to the best separation of survival in PAH patients. Table 1. Antibody kits used for ELISA.

Patients with congenital diaphragmatic hernia (CDH) often suffer from severe pulmonary hypertension, and the choice of current vasodilator therapy is mostly based on trial and error. Because pulmonary vascular abnormalities are already present early during development, we performed a study to modulate these pulmonary vascular changes at an early stage during gestation. Pregnant Sprague-Dawley rats were treated with nitrofen at day 9.5 of gestation (E9.5) to induce CDH in the offspring, and subsequently, the phosphodiesterase-5 inhibitor sildenafil and/or the novel prostaglandin-I receptor agonist selexipag (active compound NS-304) were administered from E17.5 until E20.5. The clinical relevant start of the treatment corresponds to week 20 of gestation in humans, when CDH is usually detected by ultrasound. CDH pups showed increased density of air saccules that was reverted after the use of only sildenafil. The pulmonary vascular wall was thickened, and right ventricular hypertrophy was present in the CDH group and improved both after single treatment with sildenafil or selexipag, whereas the combination therapy with both compounds did not have additive value. In conclusion, antenatal treatment with sildenafil improved airway morphogenesis and pulmonary vascular development, whereas selexipag only acted positively on pulmonary vascular development. The combination of both compounds did not act synergistically, probably because of a decreased efficiency of both compounds caused by cytochrome- P450 3A4 interaction and induction. These new insights create important possibilities for future treatment of pulmonary vascular abnormalities in CDH patients already in the antenatal period of life.

Aims Formation of a functional vascular system is essential and its formation is a highly regulated process initiated during embryogenesis, which continues to play important roles throughout life in both health and disease. In previous studies, Fzd5 was shown to be critically involved in this process and here we investigated the molecular mechanism by which endothelial loss of this receptor attenuates angiogenesis. Methods and results Using short interference RNA-mediated loss-of-function assays, the function and mechanism of signaling via Fzd5 was studied in human endothelial cells (ECs). Our findings indicate that Fzd5 signaling promotes neovessel formation in vitro in a collagen matrix-based 3D co-culture of primary vascular cells. Silencing of Fzd5 reduced EC proliferation, as a result of G0/G1 cell cycle arrest, and decreased cell migration. Furthermore, Fzd5 knockdown resulted in enhanced expression of the factors Angpt2 and Flt1, which are mainly known for their destabilizing effects on the vasculature. In Fzd5-silenced ECs, Angpt2 and Flt1 upregulation was induced by enhanced PKC signaling, without the involvement of canonical Wnt signaling, non-canonical Wnt/Ca2+-mediated activation of NFAT, and non-canonical Wnt/PCP-mediated activation of JNK. We demonstrated that PKC-induced transcription of Angpt2 and Flt1 involved the transcription factor Ets1. Conclusions The current study demonstrates a pro-angiogenic role of Fzd5, which was shown to be involved in endothelial tubule formation, cell cycle progression and migration, and partly does so by repression of PKC/Ets1-mediated transcription of Flt1 and Angpt2. Electronic supplementary material The online version of this article (10.1007/s10456-018-9625-6) contains supplementary material, which is available to authorized users.

Spondyloartritis (SpA) is een verzamelnaam voor reumatische aandoeningen waarbij ontstekingen aanwezig zijn in het bekken en de wervelkolom. Tegenwoordig wordt er een onderscheid gemaakt tussen axiale SpA en perifere SpA. Bij axiale SpA overheersen symptomen van het axiale skelet, de wervelkolom en het bekken (sacroiliitis, spondylitis). Ankyloserende spondylitis (de ziekte van Bechterew) is de bekendste vorm van axiale spondyloartritis.

Group3 medulloblastoma (MBG3) that predominantly occur in young children are usually associated with MYC amplification and/or overexpression, frequent metastasis and a dismal prognosis. Physiologically relevant MBG3 models are currently lacking, making inferences related to their cellular origin thus far limited. Using in utero electroporation, we here report that MBG3 mouse models can be developed in situ from different multipotent embryonic cerebellar progenitor cells via conditional expression of Myc and loss of Trp53 function in several Cre driver mouse lines. The Blbp-Cre driver that targets embryonic neural progenitors induced tumors exhibiting a large-cell/anaplastic histopathology adjacent to the fourth ventricle, recapitulating human MBG3. Enforced co-expression of luciferase together with Myc and a dominant-negative form of Trp53 revealed that GABAergic neuronal progenitors as well as cerebellar granule cells give rise to MBG3 with their distinct growth kinetics. Cross-species gene expression analysis revealed that these novel MBG3 models shared molecular characteristics with human MBG3, irrespective of their cellular origin. We here developed MBG3 mouse models in their physiological environment and we show that oncogenic insults drive this MB subgroup in different cerebellar lineages rather than in a specific cell of origin.

BACKGROUND Embryonal tumours with multilayered rosettes (ETMR) are highly aggressive tumors, mostly occurring in infants. Published clinical data refer to retrospective cohorts of inhomogeneously treated patients. Here, we describe the outcome of patients, who were prospectively treated within the P-HIT2000-trial, the subsequent HIT2000-interim-registry and earlier HIT-trials. PATIENTS AND METHODS Nineteen patients from the P-HIT2000-trial (2001–2011), 12 patients from the subsequent HIT2000-interim-registry (2012–2014) and 4 patients from earlier HIT-trials with centrally reviewed neuropathological and molecularly-confirmed diagnosis of ETMR were included. Outcome of 18 patients treated with carboplatin-etoposide-induction followed by tandem-high-dose chemotherapy (“CARBO-ETO+HDCT”) with stage-stratified radiotherapy administered in case of persistant disease, relapse or progression were compared to patients treated with HIT-SKK chemotherapy ± radiotherapy (n=9) or other regimens (n=8). RESULTS Median age at diagnosis was 2.9(1.0–5.3) years. Metastases at diagnosis were detected in 9 patients (26%). For the entire cohort of n=35, 5-year overall survival (OS) was 26.7%, and progression-free survival (PFS) was 18.5%. Five-year OS for patients with CARBO-ETO+HDCT, SKK chemotherapy or other regimens was 44.4%, 13.0% and 0%, respectively (p=0.006). Five-year PFS was 33.3%, 0% and 0%, respectively (p=0.119). Of 10 survivors, n=8 were treated with CARBO-ETO+HDCT; n=4 had craniospinal, n=2 local and n=4 no radiotherapy. Impact of initial gross-total-resection (p=0.231) and non-metastatic disease (p=0.097) was limited. CONCLUSIONS We show improved survival with carboplatin-etoposide-induction followed by tandem-high-dose chemotherapy, indicating that a cure is possible for some patients. However, despite intensive treatment, outcome is unsatisfactory and innovative therapies urgently need to be included in an upfront setting.

Aims: DNA methylation-based central nervous system (CNS) tumour classification has identified numerous molecularly distinct tumour types, and clinically relevant subgroups among known CNS tumour entities that were previously thought to represent homogeneous diseases. Our study aimed at characterizing a novel, molecularly defined variant of glioneuronal CNS tumour. Patients and methods: DNA methylation profiling was performed using the Infinium MethylationEPIC or 450 k BeadChip arrays (Illumina) and analysed using the ‘conumee’ package in R computing environment. Additional gene panel sequencing was also performed. Tumour samples were collected at the German Cancer Research Centre (DKFZ) and provided by multinational collaborators. Histological sections were also collected and independently reviewed. Results: Genome-wide DNA methylation data from >25 000 CNS tumours were screened for clusters separated from established DNA methylation classes, revealing a novel group comprising 31 tumours, mainly found in paediatric patients. This DNA methylation-defined variant of low-grade CNS tumours with glioneuronal differentiation displays recurrent monosomy 14, nuclear clusters within a morphology that is otherwise reminiscent of oligodendroglioma and other established entities with clear cell histology, and a lack of genetic alterations commonly observed in other (paediatric) glioneuronal entities. Conclusions: DNA methylation-based tumour classification is an objective method of assessing tumour origins, which may aid in diagnosis, especially for atypical cases. With increasing sample size, methylation analysis allows for the identification of rare, putative new tumour entities, which are currently not recognized by the WHO classification. Our study revealed the existence of a DNA methylation-defined class of low-grade glioneuronal tumours with recurrent monosomy 14, oligodendroglioma-like features and nuclear clusters.

A growing number of people desire ART with cryopreserved donor oocytes. The allocation of these oocytes to couples and mothers to be is a 2-fold process. The first step is to select a pool of recipients. The second step is to decide who should be treated first. Prioritizing recipients is critical in settings where demand outstrips supply. So far, the issue of how to fairly allocate cryopreserved donor oocytes has been poorly addressed. Our ethical analysis aims to support clinics involved in allocation decisions by formulating criteria for recipient selection irrespective of supply (Part I) and recipient prioritization in case supply is limited (Part II). Relevant criteria for recipient selection are: a need for treatment to experience parenthood; a reasonable chance for successful treatment; the ability to safely undergo an oocyte donation pregnancy; and the ability to establish a stable and loving relationship with the child. Recipients eligible for priority include those who: have limited time left for treatment; have not yet experienced parenthood; did not undergo previous treatment with cryopreserved donor oocytes; and contributed to the supply of donor oocytes by bringing a donor to the bank. While selection criteria function as a threshold principle, we argue that the different prioritization criteria should be carefully balanced. Since specifying and balancing the allocation criteria undoubtedly raises a moral dispute, a fair and legitimate allocation process is warranted (Part III). We argue that allocation decisions should be made by a multidisciplinary committee, staffed by relevant experts with a variety of perspectives. Furthermore, the committees’ reasoning behind decisions should be transparent and accessible to those affected: clinicians, donors, recipients and children born from treatment. Insight into the reasons that underpin allocation decisions allows these stakeholders to understand, review and challenge decisions, which is also known as accountability for reasonableness.

Rhabdoid tumors (RTs) are pediatric cancers that account for nearly 10% of all tumors in infants. Currently, RTs are classified according to tumor location. In the central nervous system, they are called atypical teratoid/rhabdoid tumors (ATRTs) as compared to extra-cranial malignant RTs (MRTs). Regardless of the site of occurrence, RTs share histological characteristics and genetic alterations, with >95% of them showing loss of SMARCB1, a subunit of the chromatin-remodeling SWI/SNF complex. Molecularly, RTs are heterogeneous. Previous studies reported molecular subgroups within RTs of the same anatomic compartment. However, biological similarities among RT subgroups of different sites, and molecular characteristics shared among them remained unknown. To investigate the similarity of RTs from different anatomic sites at a molecular level, we compared DNA methylation, gene expression, and H3K27ac profiles of 150 MRTs and 161 ATRTs generated by WGBS, 450K/850K arrays, RNA-, and ChIP-Seq. Unsupervised clustering of DNA methylation data showed that a subset of MRTs clustered with the MYC-subgroup of ATRTs but not with SHH- and TYR-subgroups of ATRTs. Over-expression of MYC-ATRT representative genes, such as c-MYC and the non-coding regulatory RNA gene HOTAIR, was also observed in these MRTs. Furthermore, they were both characterized by global hypomethylation, high prevalence of partially methylated domains, and similar super-enhancer regions, identified by H3K27ac ChIP-seq analyses, such as those for the HOXC cluster genes. Our results indicate that MYC-ATRTs and a subset of MRTs share molecular similarities and associate with distinct developmental processes compared to other ATRT and MRT subgroups.

INTRODUCTION: Children with high-risk medulloblastoma, supratentorial primitive neuroectodermal tumor (CNS-PNET) and pineoblastoma (PBLs) have poor survival and were enrolled in this prospective trial. However, subsequent molecular profiling uncovered diagnostic discrepancies not detectable at protocol inception. Outcomes for patients with CNS-PNET/PBL are reported here. METHODS: In this phase 3 trial, children aged 3–22 years with newly-diagnosed CNS-PNET/PBL were randomly assigned to receive carboplatin during radiation and/or adjuvant isotretinoin after standard intensive therapy. DNA methylation profiling was retrospectively completed in patients with sufficient tissue. RESULTS: Out of eighty-five participants with institutionally-diagnosed CNS-PNETs/PBLs, 60 patients underwent molecular analysis. 31/60 were non-pineal in location, of which 22 (71%) represented tumors not intended for trial inclusion, including 18 high-grade gliomas (HGG), two atypical teratoid rhabdoid tumors, and two ependymomas. Retrospective central review was more consistent with molecular diagnosis, but maintained the discrepancy in some cases. Outcomes across tumor types were strikingly different. Patients with supratentorial embryonal tumors/PBLs exhibited 5-year event-free (EFS) and overall survival (OS) of 62.8% (95%CI,43.4%-82.2%) and 78.5% (95%CI,62.2–94.8%), respectively, while patients with molecularly-classified HGG had EFS/OS of 5.6% (95%CI,0%-13.0%) and 12.0% (95%CI,0%-24.7%), respectively. Neither carboplatin nor isotretinoin significantly altered outcomes. Survival for patients with HGG was similar to historic studies without craniospinal irradiation or intensive chemotherapy. CONCLUSIONS: For patients with CNS-PNETs/PBLs, prognosis is considerably better than previously assumed when molecularly-confirmed HGG are removed. Identification of HGG may spare children from unhelpful intensive treatment. This trial highlights the challenges of histology-based diagnosis and indicates that molecular profiling should become a standard component of initial diagnosis.

Pilocytic astrocytoma (PA) is the most common brain tumor in children but is rare in adults, and hence poorly studied in this age group. We investigated 222 PA and report increased aneuploidy in older patients. Aneuploid genomes were identified in 45% of adult compared with 17% of pediatric PA. Gains were non-random, favoring chromosomes 5, 7, 6 and 11 in order of frequency, and preferentially affecting non-cerebellar PA and tumors with BRAF V600E mutations and not with KIAA1549-BRAF fusions or FGFR1 mutations. Aneuploid PA differentially expressed genes involved in CNS development, the unfolded protein response, and regulators of genomic stability and the cell cycle (MDM2, PLK2),whose correlated programs were overexpressed specifically in aneuploid PA compared to other glial tumors. Thus, convergence of pathways affecting the cell cycle and genomic stability may favor aneuploidy in PA, possibly representing an additional molecular driver in older patients with this brain tumor.

Sort strategy for CD45+/Col-1+ cells. (A) The conventional strategy with the CD45+/Col-1+ cells in red. (B) Position of the same fibrocytes when employing a gating strategy based on additional extracellular markers. The sort strategy is based on extracellular markers and with some modifications previously published [16] (of note: for our research question we did not exclude SSChi cells). CD45+ cells were analyzed for HLA-DR expression and lineage markers to exclude B-cells (CD19), NK cells (CD56) and T-cells (CD3). Lineage negative cells were plotted as CD14 versus CD16 to create a distinct group of cells enriched for CD45+/Col-1+ cells (red box). (PDF 75 kb)

Characteristics of patients and the healthy subjects used for experiments. Data are mean ± standard deviation, unless indicated otherwise. a 1 patient with chronic thromboembolic PH and 4 patients with PH secondary to an auto-immune disease. b 6 patients with IPF, 3 patients with UIP pattern secondary to extrinsic allergic alveolitis, 2 patients with an non-specific interstitial pneumonia and 1 patient with anti-synthetase syndrome. c Mean FVC/FEV1 ratio = 0.71 (2 out of 9 had an obstructive pulmonary function test, both classified as GOLD A). d Assessed by right heart catheterization or suspected with echocardiography. IPF = idiopathic pulmonary fibrosis, IPAH = idiopathic pulmonary arterial hypertension, FVC = forced vital capacity, TLCO = diffusing capacity for carbon monoxide, PAP = pulmonary arterial pressure, RAP = right atrium pressure, Svo2 = mixed venous saturation, PDE5i = phosphodiesterase type 5 inhibitor, ERA = endothelin receptor antagonist, Pros-A = prostacyclin agonist (DOCX 22 kb)

Complete overview collagen-1 and CD15 staining on CD45+/Col-1+ cells and controls. Immunocytochemical images of cultured fibroblasts, cultured fibrocytes, sorted classical monocytes and sorted CD45+/Col-1+ cells. Indicated cells were stained with Collagen-1 or isotype control (rabbit IgG) and CD15. As a control for CD15 we used a buffy coat, nicely showing positive granulocytes next to negative lymphocytes. Magnification for all images was 100Ă . (PDF 1282 kb)

Congenital diaphragmatic hernia (CDH) is a rare congenital anomaly characterised by a diaphragmatic defect, persistent pulmonary hypertension (PH) and lung hypoplasia. The relative contribution of these three elements can vary considerably in individual patients. Most affected children suffer primarily from the associated PH, for which the therapeutic modalities are limited and frequently not evidence based. The vascular defects associated with PH, which is characterised by increased muscularisation of arterioles and capillaries, start to develop early in gestation. Pulmonary vascular development is integrated with the development of the airway epithelium. Although our knowledge is still incomplete, the processes involved in the growth and expansion of the vasculature are beginning to be unravelled. It is clear that early disturbances of this process lead to major pulmonary growth abnormalities, resulting in serious clinical challenges and in many cases death in the newborn. Here we provide an overview of the current molecular pathways involved in pulmonary vascular development. Moreover, we describe the abnormalities associated with CDH and the potential therapeutic approaches for this severe abnormality.

Circulating fibrocyte numbers in patients with IPF and idiopathic pulmonary hypertension (IPAH). (A) Absolute numbers of circulating fibrocytes per ml blood in frozen PBMC of HC, patients with IPF and patients with IPAH. ** PÂ

Correlation circulating CD45+/Col-1+ fibrocytes and granulocytes. For this experiment we analyzed paired total white blood cells (open dots) and PBMCs (black dots) on the same day as blood withdrawal of 9 patients (4 IPF patients and 5 PH patients). Correlation coefficients were calculated using Spearmanâ s rank method. (PDF 102 kb)

Background The demand for donor oocytes has increased dramatically over the years. Today people in need of ART with the use of donor oocytes can appeal to commercial or public donor oocyte banks. The introduction of oocyte banks has shed a new light on the practice of ART using donor oocytes. The establishment and maintenance of oocyte banks should be sensitive to the ethical considerations. However, it is currently unclear which ethical aspects have to be taken into account. Objective and rationale The aim of this article is to identify the ethical aspects of establishing and maintaining oocyte banks for third-party ART. Search methods A systematic search was performed in July 2016 and February 2017 in both PubMed and Embase using a search string that combined synonyms for oocytes, donation or banking, reproductive care and ethics. We included a wide variety of English-language articles with a reasoned description of ethical aspects or moral considerations on oocyte donation or banking for third-party ART. Outcomes The practice of oocyte banking consists of three components, namely, the intake, storage and distribution of donor oocytes, and each is associated with multiple ethical challenges. The majority of the literature discusses ethical aspects with regard to the intake of donor oocytes, taking into account both the interests of the donor and those of the potential child. Ethical aspects related to the donor are the risks and psychosocial impact of donation, motivations and compensation in donor recruitment, and requirements for informed consent. Ethical aspects related to the potential child are 2-fold: first, the welfare standard and the selection of donors, and second, anonymity and disclosure. Ethical aspects of storing donor oocytes for ART are quality standards, confidentiality, issues of ownership and control, and international transport of donor oocytes. Ethical aspects of the distribution of donor oocytes concern the selection of recipients and the acceptability of treatment of 'non-traditional' families in particular, prioritization of recipients in case of scarcity, cross-border reproductive care, matching of recipients and donor oocytes, informed consent and counselling for recipients. Wider implications Our review demonstrates that multiple ethical aspects have to be taken into account when establishing and maintaining an oocyte bank. Yet, for many of these aspects there is no consensus regarding what approach should be employed. Remarkably, the existing literature focuses mainly on ethical aspects related to the intake of donor oocytes, while aspects related to storage and distribution of donor oocytes are less often addressed. An important gap in the existing literature should therefore be acknowledged. To conclude, our findings can serve as a starting point for clinicians in the field of ART, to conceptualize what challenges arise when establishing and maintaining oocyte banks for third-party ART. The review may also stimulate policy makers to set up a trustworthy and adaptive governance structure for the intake, storage and distribution of donor oocytes.

Pulmonary vascular diseases of the newborn comprise a wide range of pathological conditions with developmental abnormalities in the pulmonary vasculature. Clinically, pulmonary arterial hypertension (PH) is characterized by persistent increased resistance of the vasculature and abnormal vascular response. The classification of PH is primarily based on clinical parameters instead of morphology and distinguishes five groups of PH. Congenital lung anomalies, such as alveolar capillary dysplasia (ACD) and PH associated with congenital diaphragmatic hernia (CDH), but also bronchopulmonary dysplasia (BPD), are classified in group three. Clearly, tight and correct regulation of pulmonary vascular development is crucial for normal lung development. Human and animal model systems have increased our knowledge and make it possible to identify and characterize affected pathways and study pivotal genes. Understanding of the normal development of the pulmonary vasculature will give new insights in the origin of the spectrum of rare diseases, such as CDH, ACD, and BPD, which render a significant clinical problem in neonatal intensive care units around the world. In this review, we describe normal pulmonary vascular development, and focus on four diseases of the newborn in which abnormal pulmonary vascular development play a critical role in morbidity and mortality. In the future perspective, we indicate the lines of research that seem to be very promising for elucidating the molecular pathways involved in the origin of congenital pulmonary vascular disease.

Screening of eight carbohydrate acetyl esterases for their activity towards xanthan resulted in the recognition of one active esterase. AXE3, a CAZy family CE1 acetyl xylan esterase originating from Myceliophthora thermophila C1, removed 31% of all acetyl groups present in xanthan after a 48 h incubation. AXE3 activity towards xanthan was only observed when xanthan molecules were in the disordered conformation. Optimal performance towards xanthan was observed at 53 °C in the complete absence of salt, a condition favouring the disordered conformation. AXE3-deacetylated xanthan was hydrolyzed using cellulases and analyzed for its repeating units using UPLC–HILIC–ELSD/ESI–MS. This showed that AXE3 specifically removes the acetyl groups positioned on the inner mannose and that acetyl groups positioned on the outer mannose are not removed at all. After a prolonged incubation at optimal conditions, 57% of all acetyl groups, representing 70% of all acetyl groups on the inner mannose units, were hydrolyzed.

YAP1 fusion-positive supratentorial ependymomas predominantly occur in infants, but the molecular mechanisms of oncogenesis are unknown. Here we show YAP1-MAMLD1 fusions but not YAP1 wildtype are sufficient to drive malignant transformation of neural progenitors in the developing cerebral cortex in mice, and the resulting tumours share histo-molecular characteristics of human ependymomas. Nuclear localization of YAP1-MAMLD1 protein is associated with its oncogenicity and is mediated by the nuclear localization signal of MAMLD1 in a YAP1-Ser127 phosphorylation-independent manner. Chromatin immunoprecipitation-sequencing analyses of human YAP1-MAMLD1-positive ependymoma reveal enrichment of NFI and TEAD transcription factor binding site motifs in YAP1-bound regulatory elements, hypothesizing the important role of these transcription factors in YAP1-MAMLD1-driven tumourigenesis. Indeed, co-immunoprecipitation assays revealed physical interactions of TEADs and NFIA/B with the YAP1 and MAMLD1 domains of the fusion protein, respectively. Mutation of the TEAD binding site in the YAP1 fusion or repression of NFI targets prevents tumour induction in mice. Together, these results demonstrate that the YAP1-MAMLD1 fusion functions as an oncogenic driver of ependymoma through recruitment of TEADs and NFIs, indicating a rationale for preclinical studies to block the interaction between YAP1 fusions and NFI and TEAD transcription factors.

Atypical teratoid/rhabdoid tumors (AT/RT) are among the most common malignant brain tumors in infants. Comprehensive genomic studies revealed three distinct molecular subgroups (AT/RT-TYR, -MYC and -SHH). As primary cilia (PC) have already been shown to play a pivotal role in other tumor entities, we aimed to characterize the distribution of PC across AT/RT subgroups and to target primary ciliogenesis in these tumors with dismal prognosis. We performed immunofluorescence to detect PC in AT/RT primary tumor sections and cell lines. The functional role of PC was investigated in vitro by knockdown of KIF3A. The relevance of primary ciliogenesis in AT/RT biology was further elucidated in vivo using a fly model of SMARCB1 deficiency and an orthotopic mouse model. We detected PC in all AT/RT primary tumor sections and cell lines investigated in this study. Notably, we observed a significant subgroup-specific difference in the proportion of PC-positive cells. Specifically, AT/RT-TYR demonstrated the highest percentage of ciliated cells. Knockdown of KIF3A significantly reduced cell proliferation and stem cell properties. Additionally, apoptosis was significantly increased via upregulation of JAK1/STAT1 signaling. In a fly model of SMARCB1 deficiency, concomitant knockdown of several cilia-associated genes resulted in a substantial shift of the lethal phenotype with >20% of flies reaching adulthood. Finally, we demonstrated a significantly prolonged survival in an orthotopic xenograft model upon knockdown of KIF3A. In conclusion, our results implicate PC as a key feature of AT/RT biology and suggest primary ciliogenesis as a potential therapeutic target, especially in AT/RT-TYR.

Five xanthans were hydrolyzed to their repeating units using cellulases. Hydrophilic interaction chromatography with online electrospray ionization ion trap mass spectrometry and evaporative light scattering detection was used to analyze the oligomers released. It was concluded that six different pentamer repeating units (RUs) exists within a xanthan sample. The most abundant RU shows acetylation on the inner mannose and pyruvylation on the outer mannose. The second most abundant RU shows acetylation on both the inner and the outer mannose. It becomes clear that more variations in the xanthan structure exist than generally recognized. Comparison of five different xanthan samples revealed that, although the molecular composition of xanthan samples can be exactly the same, the ratio in which the RUs occur can differ significantly. It is, therefore, concluded that xanthan samples should be characterized for both, their molecular composition and the relative abundance of the RUs present.