Recently, we described a machine learning approach for classification of central nervous system tumors based on the analysis of genome-wide DNA methylation patterns [6]. Here, we report on DNA methylation-based central nervous system (CNS) tumor diagnostics conducted in our institution between the years 2015 and 2018. In this period, more than 1000 tumors from the neurosurgical departments in Heidelberg and Mannheim and more than 1000 tumors referred from external institutions were subjected to DNA methylation analysis for diagnostic purposes. We describe our current approach to the integrated diagnosis of CNS tumors with a focus on constellations with conflicts between morphological and molecular genetic findings. We further describe the benefit of integrating DNA copy-number alterations into diagnostic considerations and provide a catalog of copy-number changes for individual DNA methylation classes. We also point to several pitfalls accompanying the diagnostic implementation of DNA methylation profiling and give practical suggestions for recurring diagnostic scenarios.Electronic supplementary materialThe online version of this article (10.1007/s00401-018-1879-y) contains supplementary material, which is available to authorized users.

Acute generalized exanthematous pustulosis (AGEP) is an uncommon eruption most often provoked by drugs, by acute infections with enteroviruses, or by mercury. It is characterized by acute, extensive formation of nonfollicular sterile pustules on erythematous background, fever, and peripheral blood leukocytosis. We present clinical and immunological data on four patients with this disease, which is caused by different drugs. An involvement of T cells could be implied by positive skin patch tests and lymphocyte transformation tests. Immunohistochemistry revealed a massive cell infiltrate consisting of neutrophils in pustules and T cells in the dermis and epidermis. Expression of the potent neutrophil-attracting chemokine IL-8 was elevated in keratinocytes and infiltrating mononuclear cells. Drug-specific T cells were generated from the blood and skin of three patients, and phenotypic characterization showed a heterogeneous distribution of CD4/CD8 phenotype and of T-cell receptor Vβ-expression. Analysis of cytokine/chemokine profiles revealed that IL-8 is produced significantly more by drug-specific T cells from patients with AGEP compared with drug-specific T cells from patients that had non-AGEP exanthemas. In conclusion, our data demonstrate the involvement of drug-specific T cells in the pathomechanism of this rather rare and peculiar form of drug allergy. In addition, they indicate that even in some neutrophil-rich inflammatory responses specific T cells are engaged and might orchestrate the immune reaction. T cells from the circulation (19,20) and an enhanced eotaxin production by endothelial cells and infiltrating T cells (19). Interestingly, previous studies in patients with AGEP have revealed a high rate of strongly positive patch tests to drugs compared with patients with other drug eruptions (21)(22)(23)(24). This suggests that T cells are involved in AGEP as well. They may contribute to the accumulation of polymorphonuclear neutrophils (PMN) at the site of the lesions by the preferential release of PMN-attractive chemokines, mainly CXC chemokines such as 26).To understand the function of T cells in AGEP patients we generated drug-specific TCLs and TCCs from the circulation and skin biopsy specimens and analyzed their phenotypes, specificities, and cytokine and chemokine profiles in vitro. Immunohistochemical analysis of the acute and patch-test lesions ex vivo was performed to determine the nature of the inflammatory cell infiltrate in vivo and to supplement the in vitro studies. The data point to an important role of drug-specific T cells in AGEP, which clearly exert distinct functions compared with other cutaneous drug eruptions. MethodsPatients. A summary of the patients' clinical characteristics is given in Table 1. Patient AP developed a generalized erythema after oral administration of Augmentin (amoxicillin and clavulanic acid; SmithKline Beecham Pharmaceuticals, Irvine, United Kingdom), which was given to treat an otitis media. She had fever (40°C) and developed pustules on the back, arms, and f...

Protein kinase D (PKD) has been identified as a crucial regulator of secretory transport at the trans-Golgi network (TGN). Recruitment and activation of PKD at the TGN is mediated by the lipid diacylglycerol, a pool of which is generated by sphingomyelin synthase from ceramide and phosphatidylcholine. The nonvesicular transfer of ceramide from the endoplasmic reticulum to the Golgi complex is mediated by the lipid transfer protein CERT (ceramide transport). In this study, we identify CERT as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT toward its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. We also show that CERT, in turn, is critical for PKD activation and PKD-dependent protein cargo transport to the plasma membrane. Thus, the interdependence of PKD and CERT is key to the maintenance of Golgi membrane integrity and secretory transport.