Data on biomarker-assisted diagnosis of invasive aspergillosis (IA) in pediatric patients is scarce. Therefore, we conducted a cohort study over two years including 404 serum specimens of 26 pediatric patients after allogeneic hematopoietic stem cell transplantation (alloSCT). Sera were tested prospectively twice weekly for Aspergillus-specific DNA, galactomannan (GM), and retrospectively for (1→3)-β-D-glucan (BDG). Three probable IA and two possible invasive fungal disease (IFD) cases were identified using the European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSGERC) 2019 consensus definitions. Sensitivity and specificity for diagnosis of probable IA and possible IFD was 80% (95% confidential interval (CI): 28–99%) and 55% (95% CI: 32–77%) for BDG, 40% (95% CI: 5–85%) and 100% (95% CI: 83–100%) for GM, and 60% (95% CI: 15–95%) and 95% (95% CI: 75–100%) for Aspergillus-specific real-time PCR. However, sensitivities have to be interpreted with great caution due to the limited number of IA cases. Interestingly, the low specificity of BDG was largely caused by false-positive BDG results that clustered around the date of alloSCT. The following strategies were able to increase BDG specificity: two consecutive positive BDG tests for diagnosis (specificity 80% (95% CI: 56–94%)); using an optimized cutoff value of 306 pg/mL (specificity 90% (95% CI: 68–99%)) and testing BDG only after the acute posttransplant phase. In summary, BDG can help to diagnose IA in pediatric alloSCT recipients. However, due to the poor specificity either an increased cutoff value should be utilized or BDG results should be confirmed by an alternative Aspergillus assay.

A new and easy polymerase chain reaction (PCR) multiplex strategy, for the identification of the most common fungal species involved in invasive fungal infections (IFI) was developed in this work. Two panels with species-specific markers were designed, the Candida Panel for the identification of Candida species, and the Filamentous Fungi Panel for the identification of Aspergillus species and Rhizopusarrhizus. The method allowed the correct identification of all targeted pathogens using extracted DNA or by colony PCR, showed no cross-reactivity with nontargeted species and allowed identification of different species in mixed infections. Sensitivity reached 10 to 1 pg of DNA and was suitable for clinical samples from sterile sites, with a sensitivity of 89% and specificity of 100%. Overall, the study showed that the new method is suitable for the identification of the ten most important fungal species involved in IFI, not only from positive blood cultures but also from clinical samples from sterile sites. The method provides a unique characteristic, of seeing the peak in the specific region of the panel with the correct fluorescence dye, that aids the ruling out of unspecific amplifications. Furthermore, the panels can be further customized, selecting markers for different species and/or resistance genes.

Purpose Knowledge on Ruxolitinib exposure in patients with graft versus host disease (GvHD) is scarce. The purpose of this prospective study was to analyze Ruxolitinib concentrations of GvHD patients and to investigate effects of CYP3A4 and CYP2C9 inhibitors and other covariates as well as concentration-dependent effects. Methods 262 blood samples of 29 patients with acute or chronic GvHD who were administered Ruxolitinib during clinical routine were analyzed. A population pharmacokinetic model obtained from myelofibrosis patients was adapted to our population and was used to identify relevant pharmacokinetic properties and covariates on drug exposure. Relationships between Ruxolitinib exposure and adverse events were assessed. Results Median of individual mean trough serum concentrations was 39.9 ng/mL at 10 mg twice daily (IQR 27.1 ng/mL, range 5.6-99.8 ng/mL). Applying a population pharmacokinetic model revealed that concentrations in our cohort were significantly higher compared to myelofibrosis patients receiving the same daily dose (p < 0.001). Increased Ruxolitinib exposure was caused by a significant reduction in Ruxolitinib clearance by approximately 50%. Additional comedication with at least one strong CYP3A4 or CYP2C9 inhibitor led to a further reduction by 15% (p < 0.05). No other covariate affected pharmacokinetics significantly. Mean trough concentrations of patients requiring dose reduction related to adverse events were significantly elevated (p < 0.05). Conclusion Ruxolitinib exposure is increased in GvHD patients in comparison to myelofibrosis patients due to reduced clearance and comedication with CYP3A4 or CYP2C9 inhibitors. Elevated Ruxolitinib trough concentrations might be a surrogate for toxicity.

Acute myeloid leukemia (AML) is characterized by recurrent genetic events. The BCL6 corepressor (BCOR) and its homolog, the BCL6 corepressor-like 1 (BCORL1), have been reported to be rare but recurrent mutations in AML. Previously, smaller studies have reported conflicting results regarding impacts on outcomes. Here, we retrospectively analyzed a large cohort of 1529 patients with newly diagnosed and intensively treated AML. BCOR and BCORL1 mutations were found in 71 (4.6%) and 53 patients (3.5%), respectively. Frequently co-mutated genes were DNTM3A, TET2 and RUNX1. Mutated BCORL1 and loss-of-function mutations of BCOR were significantly more common in the ELN2017 intermediate-risk group. Patients harboring loss-of-function mutations of BCOR had a significantly reduced median event-free survival (HR = 1.464 (95%-Confidence Interval (CI): 1.005–2.134), p = 0.047), relapse-free survival (HR = 1.904 (95%-CI: 1.163–3.117), p = 0.01), and trend for reduced overall survival (HR = 1.495 (95%-CI: 0.990–2.258), p = 0.056) in multivariable analysis. Our study establishes a novel role for loss-of-function mutations of BCOR regarding risk stratification in AML, which may influence treatment allocation.

Antikörper, die gegen eine klinisch relevante Gruppe von Rezeptoren innerhalb der Tumornekrosefaktor-Rezeptor-Superfamilie (TNFRSF) gerichtet sind, darunter CD40 und CD95 (Fas/Apo-1), benötigen ebenfalls eine Bindung an Fc-Gamma-Rezeptoren (FcγRs), um eine starke agonistische Wirkung zu entfalten. Diese FcγR-Abhängigkeit beruht weitgehend auf der bloßen zellulären Verankerung durch die Fc-Domäne des Antikörpers und benötigt dabei kein FcγR-Signalling. Ziel dieser Doktorarbeit war es, das agonistische Potenzial von αCD40- und αCD95-Antikörpern unabhängig von der Bindung an FcγRs durch die Verankerung an Myelomzellen zu entfalten. Zu diesem Zweck wurden verschiedene Antikörpervarianten (IgG1, IgG1-N297A, Fab2) gegen die TNFRSF-Mitglieder CD40 und CD95 genetisch mit einem einzelkettig kodierten B-Zell-aktivierenden Faktor (scBaff) Trimer als C-terminale myelom-spezifische Verankerungsdomäne fusioniert, welche die Fc-Domäne-vermittelte FcγR-Bindung ersetzt. Diese bispezifischen Antikörper-scBaff-Fusionsproteine wurden in Bindungsstudien und funktionellen Assays mit Tumorzelllinien untersucht, die einen oder mehrere der drei Baff-Rezeptoren exprimieren: BaffR, Transmembran-Aktivator und CAML-Interaktor (TACI) und B-Zell-Reifungsantigen (BCMA). Zelluläre Bindungsstudien zeigten, dass die Bindungseigenschaften der verschiedenen Domänen innerhalb der Antikörper-scBaff-Fusionen gegenüber der Zielantigene vollständig intakt blieben. In Ko-Kulturversuchen von CD40- und CD95-responsiven Zellen mit BaffR-, BCMA- oder TACI-exprimierenden Verankerungszellen zeigten die Antikörper-Fusionsproteine einen starken Agonismus, während in Ko-Kulturen mit Zellen ohne Expression von Baff-interagierenden Rezeptoren nur eine geringe Rezeptorstimulation beobachtet wurde. Die hier vorgestellten αCD40- und αCD95-Antikörper-scBaff-Fusionsproteine zeigen also Myelom-spezifische Aktivität und versprechen im Vergleich zu herkömmlichen CD40- und CD95-Agonisten geringere systemische Nebenwirkungen.