ALBERT

Description: The survival of patients with acute myeloid leukemia (AML) is still poor, but recent advances including Fms-like tyrosine kinase 3 (FLT3) inhibitors are encouraging. Approximately 25 % of patients with AML carry an activating internal tandem duplication (ITD) in the juxtamembrane domain (JM) of FLT3. Additionally, around 7 % of AML patients exhibit an activating point mutation in the activation loop of the tyrosine kinase domain (TKD). ITD or TKD mutations lead to constitutive activation of the receptor and cellular transformation, and ITD mutations negatively impact prognosis. In addition to the common ITD and TKD mutations we detected for the first time deletions of 3-18 bp in the juxtamembrane domain. These deletions were detected very rarely in a large cohort of newly diagnosed AML patients (7/6843; 0.1%; Munich Leukemia Laboratory). The prognostic relevance and functional consequences of these new mutations are unknown. We first performed retroviral ectopic stable expression of two of these deletion mutants delEY (p.Glu598_Tyr599del) and delIns (p.Phe590_Asp593delinsLeuTyr) in growth factor-dependent Ba/F3 cells. Our experiments demonstrate shown that both variants lead to constitutive phosphorylation of downstream signaling pathways (PI3K/AKT, Ras/MAPK, and STAT3/5). Interestingly, phosphorylation of Y591 could only be confirmed for the ITD mutant but not the other two mutants. The inhibition of protein transport to the membrane by brefeldin A or inhibition of receptor glycosylation by tunicamycin treatment showed substantial differences in signaling between FLT3 deletion mutants and FLT3-ITD. In addition, the FLT3 deletion mutants induce growth factor independent proliferation of Ba/F3 cells as well as clonal growth in semi-solid medium without addition of cytokines in a comparable manner to FLT3-ITD. Next, we analyzed the RNA expression of the STAT5 target genes pim-1, cis, bcl-xL and mcl-1 in the mutant cell lines. Proto-oncogene pim-1 is significantly upregulated in Ba/F3 cells expressing FLT3 deletion mutants, and this effect was abrogated when cells were treated with the kinase inhibitors PKC412 (50 nM) or AC220 (10 nM) for 12 hours. While only FLT3-ITD expression led to a significant upregulation of bcl-xL and mcl-1 mRNA, there was a clear reduction of apoptotic cells by trypan blue exclusion and by annexin-V staining of both FLT3 deletion mutant and FLT3-ITD expressing Ba/F3 cells, confirming a comparable low amount of apoptotic cells. By testing different concentrations of PKC412 and AC220 (0 – 50 nM) to inhibit receptor activity, a higher sensitivity to TKI of the deletion mutants in comparison to FLT3-ITD was observed. For a better understanding of the mechanism behind the constitutive activity of the JM domain mutants, we next analyzed the x-ray structure of wild type FLT3 receptor and modeled the small deletions. The deletions are situated in the switch motif (JM-S) and the zipper motif (JM-Z) of the JM domain, suggesting a malfunction in the proper orientation and guidance to the JM hinge region between the closed and open conformation. We here describe for the first time the detection and functional characterization of FLT3 deletion mutations in AML. Whereas ITD and TKD mutations in the FLT3 receptor serve as prognostic markers in AML, the prognostic relevance of the newly detected FLT3 deletion mutations is still unclear. By analyzing phosphorylation pattern, factor independent growth of Ba/F3 cells, clonal growth, quantitative PCR of STAT5 target genes, TKI sensitivity, and the x-ray structure, our study demonstrates that the described FLT3 deletions mediate constitutive activation of the receptor tyrosine kinase most likely by disruption of receptor autoinhibition. Furthermore, this analysis offers new insights of structural necessities for autoregulation of class III RTKs. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other. Brümmendorf:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Schnittger:MLL Munich Leukemia Laboratory: Other. Koschmieder:Novartis: Consultancy, Honoraria, Other, Research Funding.

Description: Key Points Droplet digital PCR is an affordable and user-friendly method for the detection of F8 and F9 sequence variants in maternal plasma. Targeted MPS accurately determines fetal inheritance of F8 int22h-related inversions, using maternal plasma of pregnant hemophilia carriers.

Description: Hemophilia is a bleeding disorder with X-linked inheritance. Current prenatal diagnostic methods for hemophilia are invasive and pose a risk to the fetus. Cell-free fetal DNA analysis in maternal plasma provides a noninvasive mean of assessing fetal sex in such pregnancies. However, the disease status of male fetuses remains unknown if mutation-specific confirmatory analysis is not performed. Here we have developed a noninvasive test to diagnose whether the fetus has inherited a causative mutation for hemophilia from its mother. The strategy is based on a relative mutation dosage approach, which we have previously established for determining the mutational status of fetuses for autosomal disease mutations. In this study, the relative mutation dosage method is used to deduce whether a fetus has inherited a hemophilia mutation on chromosome X by detecting whether the concentration of the mutant or wild-type allele is overrepresented in the plasma of heterozygous women carrying male fetuses. We correctly detected fetal genotypes for hemophilia mutations in all of the 12 studied maternal plasma samples obtained from at-risk pregnancies from as early as the 11th week of gestation. This development would make the decision to undertake prenatal testing less traumatic and safer for at-risk families.

Description: Introduction: In a recent study, we performed autologous serum infusions to evaluate the elimination kinetics of hemostasis-related biomarkers in healthy human subjects. In order to monitor a serum-induced activation of coagulation, we measured free thrombin in the infused serum and in plasma samples taken during and after infusion, but did not detect any de novo thrombin formation [PLoS One. 2015; 10(12): e0145012]. To study if the low levels of free thrombin in the infused serum induce generation of activated protein C (APC) we additionally measured APC in samples drawn after autologous serum infusion and in vitro in a purified system. Methods: Autologous serum was infused (50 mL/30 min) into 19 healthy volunteers. Four of them were simultaneously receiving infusions of the thrombin inhibitor argatroban (1 µg/kg/min), initiated 1 h before and ceased 1 h after starting the infusion of serum. Thrombin and APC were measured in serum and in plasma samples drawn before and in 15-min intervals during the infusion of serum, using a highly-sensitive oligonucleotide-based enzyme capture assay (OECA) platform. In in vitro experiments, APC formation was induced by addition of purified thrombin or serum to buffer containing protein C and thrombomodulin in excess, and CaCl2 at physiological concentrations. The formation of APC was subsequently measured by OECA. Results: In the autologous serum median (interquartile range) concentrations of thrombin and APC were 6.68 (4.63 - 8.73) ng/mL and 9.17 (7.63 - 13.91) ng/mL, thus doses of 0.12 (0.07 - 0.15) ng/mL of thrombin and 0.16 (0.14 - 0.22) ng/mL of APC were infused per mL of the subjects' plasma volume. In the plasma of probands, that did not receive argatroban, peak thrombin levels of 0.04 (0.00 - 0.08) ng/mL were measured, indicating a rapid inactivation of thrombin by endogenous inhibitors present in the plasma. However, with 1.41 (0.76 - 2.97) ng/mL peak APC levels exceeded the infused APC doses by a multiple. This was also true for the plasma samples from the probands that received argatroban, in which peak levels of APC of 0.94 (0.79 - 1.22) ng/mL were measured despite thrombin inhibition indicated by prolongation of the aPTT of 42.9 (40.1 - 44.4) s and thrombin time of 78.3 (69.3 - 87.2) s. In the in vitro experiments addition of argatroban at the concentrations achieved in the probands completely abolished APC generation up to a thrombin concentration of 5 ng/ml. Addition of human serum as a source for thrombin in the same purified system consistently induced generation of greater amounts of APC than expected on the basis of the amount of thrombin present in the serum samples. Conclusions: The data obtained provide evidence for a thrombin-independent mechanism of APC formation. Further in vitro studies with endothelial cells are required to identify the components that are involved in this alternative way of APC generation. Disclosures Rühl: CSL Behring: Research Funding; Bayer: Consultancy, Honoraria. Müller:Novo Nordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.