Diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters (DGONC) - a molecularly defined glioneuronal CNS tumour class displaying recurrent monosomy 14.

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.

[Adult ADHD versus borderline personality disorder: criteria for differential diagnosis]. / ADHS im Erwachsenenalter versus Borderline-Persönlichkeitsstörung: Kriterien zur Differenzialdiagnostik.

The present study focuses on selected symptom criteria to distinguish between attention deficit/hyperactivity disorder (ADHD) in adults and borderline personality disorder (BPD). A sample of n = 158 subjects was examined, consisting of BPD patients (n = 37), ADHD patients (n = 58), comorbid BPD/ADHD patients (n = 19), a clinical group of patients fulfilling the diagnostic criteria of a depressive disorder (DEP; n = 22) and a non-clinical control group (KG; n = 22). Selected symptom criteria were investigated by using the German scales "Skala zur Erfassung der Impulsivität und emotionalen Dysregulation der Borderline-Persönlichkeitsstörung" (IES-27), "ADHS-Screening für Erwachsene" (ADHS-LE), "Fragebogen zu dissoziativen Symptomen" (FDS) and a scale for the assessment of paranoid and dichotomous thinking (PADI). Multivariate analyses of variance revealed that BPD patients differed significantly with respect to self-mutilating behaviour, suicidality, dissociation, paranoia and dichotomy from all other groups. The same effect was found for affect regulation. Furthermore BPD patients differed significantly from ADHD patients by a more severe impulsiveness (IES-27), but not through disturbed impulse control and disinhibition overall. Regarding mean differences between ADHD and BPD patients for attentional control, ADHD patients revealed higher scores which just missed significance. For hyperactivity no significant group differences were found which is assumed to be influenced by symptom overlap like restlessness and aversive tension. The findings suggest that BPD-specific criteria, a stronger affective dysregulation and a higher tendency for autoaggressive impulsive reactions are more selective for differential diagnosis than the core symptoms of adult ADHD. Only attentional control might be a useful criterion for differential diagnosis, which should be examined in future studies.

Phase I/II intra-patient dose escalation study of vorinostat in children with relapsed solid tumor, lymphoma or leukemia.

Members of the histone deacetylase (HDAC) family exhibit great promise as potential drug targets in pediatric tumors including neuroblastoma, medulloblastoma, ependymoma and Ewing's sarcoma. HDAC inhibitors of various structural classes have shown anti-tumoral effects in pre-clinical pediatric tumor models as single agents or in combination treatments. Suberoylanilidehydroxamic acid (SAHA=vorinostat) is the most clinical advanced compound of the class and was approved by the US FDA in October 2006 for the treatment of refractory cutaneous T-cell lymphoma. In this phase I/II trial, pediatric patients with relapsed solid tumors, lymphoma or leukemias are treated according to an individualized dose escalation concept ensuring each individual patient to receive his optimal dose with respect to toxicity and efficacy. The study is accompanied by an extensive pharmacokinetic, pharmacodynamic and biomarker program.

3D perfusion mapping and virtual surgical planning in the treatment of pediatric embryonal abdominal tumors.

INTRODUCTION: 3D imaging and surgical planning for the treatment of embryonal tumors using different techniques (CT versus MRI) are presently under discussion. Up to now, the main focus has been on visualizing the anatomy. Contrast medium dynamics have not been taken into consideration. The aim of the present study was to establish the technical means of integrating the 3D images from functional MRI data into the anatomical images and to determine clinical applications for this approach. MATERIAL AND METHODS: In 11 patients (mean age: 2.4 years) with solid tumors, 26 diagnostic MRI examinations were performed for primary diagnosis, treatment monitoring, or as part of the surgical planning. Seven children presented with neuroblastomas, three with Wilms' tumor, and one with advanced bilateral nephroblastomatosis. The MRI data were acquired using a 1.5-T system. For post-processing, we used volume rendering software, including an evaluation of perfusion. By using color-coded parametric images and integrating functional information, perfusion could be visualized and used for interactive surgical planning. Macroscopic and microscopic sections served as the gold standard for assessing tissue viability. RESULTS: We were able to integrate the dynamic data into the anatomical images for all patients. A good agreement was found between the results of surgical planning, including perfusion mapping, with the surgical site, subsequently produced macroscopic sections and the results of random microscopic examinations. CONCLUSIONS: Perfusion mapping using color-coded parametric images of pediatric abdominal tumors extends the diagnostic techniques currently available. We provide first proof of the possibility of integrating functional information into 3D MR images in children. Monitoring the treatment of nephroblastoma and surgical planning for pediatric embryonal tumors represent potential applications of this technique.

Selective inhibition of HDAC8 decreases neuroblastoma growth in vitro and in vivo and enhances retinoic acid-mediated differentiation.

For differentiation-defective malignancies, compounds that modulate transcription, such as retinoic acid and histone deacetylase (HDAC) inhibitors, are of particular interest. HDAC inhibitors are currently under investigation for the treatment of a broad spectrum of cancer diseases. However, one clinical drawback is class-specific toxicity of unselective inhibitors, limiting their full anticancer potential. Selective targeting of individual HDAC isozymes in defined tumor entities may therefore be an attractive alternative treatment approach. We have previously identified HDAC family member 8 (HDAC8) as a novel target in childhood neuroblastoma. Using small-molecule inhibitors, we now demonstrate that selective inhibition of HDAC8 exhibits antineuroblastoma activity without toxicity in two xenograft mouse models of MYCN oncogene-amplified neuroblastoma. In contrast, the unselective HDAC inhibitor vorinostat was more toxic in the same models. HDAC8-selective inhibition induced cell cycle arrest and differentiation in vitro and in vivo. Upon combination with retinoic acid, differentiation was significantly enhanced, as demonstrated by elongated neurofilament-positive neurites and upregulation of NTRK1. Additionally, MYCN oncogene expression was downregulated in vitro and tumor cell growth was markedly reduced in vivo. Mechanistic studies suggest that cAMP-response element-binding protein (CREB) links HDAC8- and retinoic acid-mediated gene transcription. In conclusion, HDAC-selective targeting can be effective in tumors exhibiting HDAC isozyme-dependent tumor growth in vivo and can be combined with differentiation-inducing agents.

Transcriptional regulation of the human replacement histone gene H3.3B.

In contrast to the cell-cycle-dependent histone genes, replacement histone genes are transcribed independently of DNA replication and their expression is upregulated during differentiation. We have investigated the transcriptional regulation of the recently characterized human replacement histone gene H3.3B. Using reporter gene assays of promoter-luciferase gene-constructs, we show that promoter activity largely depends on an intact Oct and CRE/TRE element within the proximal 145 bp of the promoter. DNase I footprinting revealed binding of proteins to a 40-bp region covering these two elements. Band shift experiments identified binding proteins as Oct-1 and factors of the CREB/ATF and AP-1 family, respectively. The unexpected transcriptional regulation of this replacement histone gene is discussed.

Tumor cell differentiation by butyrate and environmental stress.

The present study shows that stress signaling plays a role in differentiation of K562, PANC1, HT29 and HL60 tumor cells: (1) Butyrate induced differentiation in K562, PANC1, and HT29 cells can be inhibited by SB203580, a specific inhibitor of p38 stress activated protein kinase. (2) Heat shock and hyperosmolarity increase expression of differentiation markers in K562, HT29, HL60 and in K562, PANC1, and HT29 cells, respectively. (3) Conversely, environmental stress induced differentiation in K562, HT29, and PANC1 cells can be inhibited by SB203580 and quercetin, a compound with heat shock pathway inhibiting activity. (4) Butyrate and environmental stress enhance either additively or synergistically differentiation of K562, HT29, PANC1 or HL60 cells, respectively. Stress signaling pathways might be an interesting pharmacologic target for differentiation therapy of malignant disease.

Reduction of gamma-aminobutyric acid-ergic neurotransmission as a putative mechanism of radiation induced activation of the gonadotropin releasing-hormone-pulse generator leading to precocious puberty in female rats.

Brain irradiation in prepubertal children with malignomas can cause precocious puberty. A selective cranial cobalt (Co(60))-irradiation technique has been developed in rats. In two experiments early juvenile (13-15 days old) female rats received a single dose of 5 Gy or sham irradiation. At pubertal age (post-natal days 33-34) irradiated rats had higher serum estradiol and luteinizing hormone levels. In experiment 1 irradiated rats had higher gonadotropin releasing-hormone (GnRH) mRNA levels in the preoptic area compared to controls (P<0.05). In experiment 2 the release rates of gamma-aminobutyric acid (GABA) in vitro from preoptic mediobasal hypothalamic areas of irradiated rats were significantly reduced after stimulation with the GABA(A) receptor agonist muscimol (maximum values 4607+/-804 vs. 7399+/-1048 pM in controls, mean+/-SEM, P<0.05). Radiation induced central precocious puberty might be caused by damage to inhibitory GABAergic neurons leading to premature activation of the GnRH-pulse generator.

Histone gene expression and chromatin structure during spermatogenesis.

The chromatin of male germ cells is restructured throughout spermatogenesis. Analysis of differential histone protein patterns at specific stages of spermatogenesis may contribute towards an understanding of the changes in chromatin structure and function during this differentiation process. The most striking changes in histone patterns occur at the stage of pachytene spermatocytes when most of the linker H1 histones are replaced by the testis specific subtype H1t. In addition, replacement of core histone subtypes is observed at this stage. These structural changes precede the reorganization of chromatin at haploid stages when histones are replaced first by transition proteins and then by protamines.

High-dose chemotherapy (HDCT) with auto-SCT in children with atypical teratoid/rhabdoid tumors (AT/RT): a report from the European Rhabdoid Registry (EU-RHAB).

A retrospective analysis of data from the European Rhabdoid Registry (EU-RHAB) was performed to describe the outcome of children with atypical teratoid/rhabdoid tumors (AT/RT) who underwent high-dose chemotherapy (HDCT) with auto-SCT. Nineteen patients (male, n=15; median age at diagnosis 21 months) were identified. Nine patients presented with metastatic disease at diagnosis. A partial or subtotal resection was achieved in 11, a total resection in five and a biopsy in three patients. Patients received a median of six chemotherapy cycles prior to HDCT. Additional radiotherapy was performed in 14 patients (first-line, n=9; following progression, n=5). Six patients underwent tandem auto-SCT. Disease status before HDCT was CR in six, PR in eight, stable disease in two and progressive disease (PD) in two patients (data missing, n=1). With a median follow-up of 16 months, 14 patients progressed. Estimated progression-free and OS at 2 years were 29% (±11%) and 50% (±12%), respectively. At last follow-up, eight patients were alive (first CR, n=4; second CR, n=2; PR, n=1; PD, n=1). Eleven patients died of PD. Median time-to-progression was 14 months. Selected patients with AT/RT might benefit from HDCT with radiotherapy. The definitive impact of this treatment modality has to be evaluated prospectively in a randomized trial.

Neuroblastoma imaging.

Neuroblastoma is an embryonic tumor of the sympathetic nervous system which represents one of the most common malignancies in early childhood. Its clinical and biological behavior show a remarkable heterogeneity, ranging from spontaneous regression to inexorable progression with a fatal outcome. This review summarizes the clinical risk stratification and treatment options. An extensive overview of the role of imaging during the course of the disease and typical imaging findings in all imaging modalities are demonstrated.

Targeting histone deacetylases in neuroblastoma.

Histone deacetylases (HDACs) are an emerging class of novel anti-cancer drug targets. Recently, studies in adult cancers and in neuroblastoma have shown that individual HDAC family members are aberrantly expressed in tumors and correlate with disease stage and prognosis. In neuroblastoma, knockdown of individual HDAC family members causes distinct phenotypes ranging from differentiation to apoptosis. HDACs are involved in controlling MYCN function and are upregulated in chemotherapy-resistant neuroblastoma cells. Treatment with unselective pan-HDAC inhibitors causes cell cycle arrest, differentiation, apoptosis, and inhibition of clonogenic growth of neuroblastoma cells, and restores susceptibility to chemotherapy treatment. The molecular mechanisms mediating the anti-cancer effects of HDAC inhibitors on neuroblastoma cells are incompletely understood and involve targeting of aberrant epigenetic repression of tumor suppressor genes, activation of developmental differentiation pathways, as well as changing the acetylation level and function of non-histone proteins. In neuroblastoma mouse models, unselective HDAC inhibitors demonstrate anti-tumoral effects. First phase I clinical trials in children with refractory cancers using HDAC inhibitors depsipeptide and the recently approved vorinostat are underway. This review summarizes our current knowledge about classical HDAC family members as novel drug targets for neuroblastoma therapy and discusses the potential role of next generation, selective HDAC inhibitors.

The epigenetics of cancer in children.

Malignant tumors of childhood represent a rather heterogeneous group of neoplasms originating from virtually any anatomical structure. Despite major improvements in the clinical management including timely diagnosis, advanced supportive care and refined multimodality treatment, prognosis remains grim for certain risk groups. Aberrant epigenetic regulation, i.e. changes in gene transcription not due to DNA sequence alterations, is now increasingly recognized as a fundamental process in malignant transformation, tumor progression and drug resistance. The molecular mechanisms involve aberrant activity of enzymes controlling the packaging and transcriptional regulation of the genome. Two major protein families are involved in this process, DNA methyltransferases and histone deacetylases. With the availability of small molecule inhibitors targeting the aberrant epigenetic machinery in cancer cells, these compounds are evaluated in several clinical trials.

Neonatal-onset multisystem inflammatory disease (NOMID) due to a novel S331R mutation of the CIAS1 gene and response to interleukin-1 receptor antagonist treatment.

Neonatal-onset multisystem inflammatory disease (NOMID) is due to mutations in the CIAS1 gene. We describe the case of a 5-year-old boy with neonatal onset of urticaria-like rash, chronic fever, laboratory findings of systemic inflammation, hepatosplenomegaly, and chronic CNS inflammation associated with sensorineural deafness. Sequence analysis of exon 3 of the CIAS1 gene revealed a novel C1754A/S331R mutation. Since experimental evidence suggests that patients with cryopyrin-associated periodic syndromes (CAPS) could respond to inhibition of binding of interleukin IL-1alpha and IL-1beta to the IL-1 receptor type 1, we treated the child with the IL-1 receptor antagonist anakinra. A remarkable clinical and serological response to therapy was observed, suggesting that pharmacological inhibition of the IL-1 signaling pathway offers an important new treatment option for patients with NOMID.

Novel valproic acid derivatives with hemoglobin F inducing activity.

Pharmacological induction of hemoglobin F expression may be a promising approach for the treatment of beta-thalassemia and sickle cell disease. Valproic acid, a drug frequently used for the treatment of seizure disorders, has been shown to enhance fetal hemoglobin synthesis in erythroid cells. However, this effect is only modest and requires relative high concentrations. Therefore, the drug appears not to be applicable for the treatment of beta-globin chain disorders. Here, we describe the identification of novel valproic acid derivatives with potent hemoglobin F inducing activities at concentrations that presumably can be obtained in vivo.

Influence of membrane-associated heparan sulfate on the internalization of the small proteoglycan decorin.

The small dermatan sulfate proteoglycan decorin is efficiently internalized by a variety of cells of mesenchymal origin. Previous studies had implicated the involvement of 51- and 26-kDa receptor proteins in this uptake process. The surface localization of these proteins has now been demonstrated by labeling with a membrane-impermeant, biotinylating reagent. The human keratinocyte cell line HaCaT exhibited only about 5% of the clearance rate of fibroblasts for exogeneously added decorin, although it was not deficient in the 51- and 26-kDa proteins. Evidence is presented that plasma membrane-associated heparan sulfate influences receptor trafficking and contributes to the low internalization rate of the receptors in keratinocytes: (i) Heparitinase digestion of intact keratinocytes led to an approximately 10-fold increase in the efficiency of decorin endocytosis. (ii) Endocytosis of decorin was increased more than 10-fold in keratinocytes in the presence of protamine, a cationic, heparan sulfate-binding protein. This effect is considered to be caused by competition between protamine and the endocytosis receptor for cell surface-associated heparan sulfate. (iii) Preincubation of keratinocytes with heparan sulfate-degrading enzymes at 37 degrees C led to a decrease of receptor proteins localized at the cell surface as judged by subsequent surface labeling at 0 degree C. (iv) An alteration of the biosynthesis of heparan sulfate proteoglycans by p-nitrophenyl-beta-xyloside was accompanied by an increased yield of intracellularly located receptor proteins. Plasma membrane-associated heparan sulfate from keratinocytes differed from the corresponding species of fibroblasts in quantity and quality. It is, therefore, suggested that the intracellular trafficking of the decorin receptor proteins is influenced by the amount and/or the composition of membrane-associated heparan sulfate.

Butyrate-induced erythroid differentiation of human K562 leukemia cells involves inhibition of ERK and activation of p38 MAP kinase pathways.

Butyrate induces cytodifferentiation in many tumor cells of different origin, suggesting that an as yet unidentified common mechanism inherent to malignant cells is the target of butyrate action. This study determined the role of different mitogen-activated protein (MAP) kinase signal transduction pathways in butyrate-induced erythroid differentiation of K562 human leukemia cells. Using a panel of anti-ERK, JNK, and p38 phosphospecific antibodies, the study showed that phosphorylation of ERK and JNK is decreased following treatment of cells with butyrate, whereas phosphorylation of p38 is increased. In contrast, a K562 subline defective in butyrate-mediated induction of erythroid differentiation did not reveal these changes in phosphorylation patterns. Inhibition of ERK activity by UO126 induces erythroid differentiation and acts synergistically with butyrate on hemoglobin synthesis and inhibition of cell proliferation, whereas inhibition of p38 activity by SB203580 completely abolished induction of hemoglobin expression by butyrate. Taken together, our data suggest a model in which butyrate induces erythroid differentiation of K562 cells by inhibition of ERK and activation of p38 signal transduction pathways.

cAMP/phorbol ester response element is involved in transcriptional regulation of the human replacement histone gene H3.3B.

The human histone H3.3B gene belongs to the group of replacement histone genes, which are up-regulated during differentiation of cells. Here we provide evidence that a cAMP response element/PMA response element (CRE/TRE) located in the proximal promoter contributes to the expression of the H3.3B gene. (1) Band shift and supershift analysis demonstrated the binding of AP-1 and transcription factors of the CRE-binding protein/activating-transcription-factor family to the H3.3B CRE/TRE. (2) Treatment of HeLa cells with PMA led to a 4-fold increase in H3. 3B mRNA levels within 2 h, whereas transcription of the cell cycle-dependent H3 histone genes remained constant. In contrast with PMA, cAMP did not affect H3.3B transcription. (3) PMA treatment of cells transiently transfected with H3.3B promoter constructs linked to a luciferase gene caused a 4-5-fold increase in reporter gene activity, whereas mutation of the CRE/TRE element abolished the PMA response. These results demonstrate that activation of the protein kinase C pathway by PMA results in an early up-regulation of H3.3B gene expression via the CRE/TRE element. Furthermore treatment with PMA apparently leads to differential induction of H3 histone subtype genes and this in turn can result in a remodelling of chromatin structure of cells before or during differentiation processes.