Comprehensive cancer predisposition testing within the prospective MASTER trial identifies hereditary cancer patients and supports treatment decisions for rare cancers.

BACKGROUND: Germline variant evaluation in precision oncology opens new paths toward the identification of patients with genetic tumor risk syndromes and the exploration of therapeutic relevance. Here, we present the results of germline variant analysis and their clinical implications in a precision oncology study for patients with predominantly rare cancers. PATIENTS AND METHODS: Matched tumor and control genome/exome and RNA sequencing was carried out for 1485 patients with rare cancers (79%) and/or young adults (77% younger than 51 years) in the National Center for Tumor Diseases/German Cancer Consortium (NCT/DKTK) Molecularly Aided Stratification for Tumor Eradication Research (MASTER) trial, a German multicenter, prospective, observational precision oncology study. Clinical and therapeutic relevance of prospective pathogenic germline variant (PGV) evaluation was analyzed and compared to other precision oncology studies. RESULTS: Ten percent of patients (n = 157) harbored PGVs in 35 genes associated with autosomal dominant cancer predisposition, whereof up to 75% were unknown before study participation. Another 5% of patients (n = 75) were heterozygous carriers for recessive genetic tumor risk syndromes. Particularly, high PGV yields were found in patients with gastrointestinal stromal tumors (GISTs) (28%, n = 11/40), and more specifically in wild-type GISTs (50%, n = 10/20), leiomyosarcomas (21%, n = 19/89), and hepatopancreaticobiliary cancers (16%, n = 16/97). Forty-five percent of PGVs (n = 100/221) supported treatment recommendations, and its implementation led to a clinical benefit in 40% of patients (n = 10/25). A comparison of different precision oncology studies revealed variable PGV yields and considerable differences in germline variant analysis workflows. We therefore propose a detailed workflow for germline variant evaluation. CONCLUSIONS: Genetic germline testing in patients with rare cancers can identify the very first patient in a hereditary cancer family and can lead to clinical benefit in a broad range of entities. Its routine implementation in precision oncology accompanied by the harmonization of germline variant evaluation workflows will increase clinical benefit and boost research.

Pharmacodynamic and safety results of PEG-Hirudin in healthy volunteers.

PEG-Hirudin, a chemically defined conjugate of recombinant hirudin and two molecules of polyethylene glycol (PEG)-5000 is a highly selective direct thrombin inhibitor with a significantly longer duration of action than non-conjugated recombinant hirudin permitting once daily subcutaneous administration. A series of placebo-controlled, randomized, Phase I clinical trials were conducted in 75 healthy volunteers to investigate the anticoagulant effects, safety and pharmacodynamics of PEG-Hirudin when administered intravenously as a bolus injection, infusion, and subcutaneously. After single i.v. injections of various doses of PEG-Hirudin (0.03-0.3 mg/kg) dose-dependent increases in anti-IIa activity and APTT were observed. Four hours after injection of 0.3 mg/kg, mean plasma concentration expressed in terms of anti-IIa activity was still 0.89 micrograms/ml, corresponding to a 1.8-fold prolongation of APTT. Continuous intravenous infusions of 0.01 and 0.02 mg/kg/h PEG-Hirudin resulted in maximum anti-IIa activities of 0.42 micrograms/ml and 0.77 micrograms/ml, respectively, at the end of a six-hour infusion period without having reached steady state at this time. After termination of the infusion, anticoagulant activity displayed an immediate exponential decrease. The anticoagulant activities of single subcutaneous doses of 0.05 to 0.6 mg/kg were studied in a further series of investigations and slow increases and prolonged durations of anti-IIa activity and APTT prolongation were found. Repeated, once daily subcutaneous administrations of 0.2 to 0.4 mg/kg for five days resulted in dose-dependent prolongations of APTT and increases in anti-IIa activity without completely reaching steady state conditions. In a further study, 0.3 mg/kg of PEG-Hirudin was given as an i.v. bolus injection followed by three repeated single daily s.c. injections. In this trial, the APTT was shorter than expected from previous studies; therefore, a direct comparison of various APTT reagents was made in the intravenous infusion trial. Of the APTT reagents tested, BioMérieux Silimat and IL-ellagic acid proved to be the most sensitive to PEG-Hirudin. The hirudin derivative PEG-Hirudin was tolerated very well without immuno-allergic side effects. In view of the significantly prolonged anticoagulant efficacy in comparison to non-conjugated r-hirudin, PEG-Hirudin is a promising compound especially for repeated once daily subcutaneous administration.

Dexverapamil as resistance modifier in acute myeloid leukaemia.

In order to evaluate dexverapamil as a resistance modifier in acute myeloid leukaemia, we have added dexverapamil (4 x 300 mg/d orally) to DA chemotherapy (daunorubicin, cytosine arabinoside) in six patients with acute myeloid leukaemia. Two patients (1 first and 1 second relapse) achieved complete remission and two patients (1 refractory disease, 1 third relapse) showed some improvement. One patient in first relapse died due to disease progression and one drug-refractory patient remained refractory. The peak plasma levels of dexverapamil and nordexverapamil ranged from about 600 to 4100 ng/ml and from 450 to 1130 ng/ml, respectively. Major sideeffects were hypotension and sinus bradycardia. These results show the need for further evaluation of dexverapamil as a resistance modifier in acute myeloid leukaemia.

Influence of palmitate and oleate on the binding of warfarin to human serum albumin: stopped-flow studies.

The rate of transition from an unstable to a stable complex and the dependence of this on the number of fatty acid ligands present was determined for the binding of warfarin on human serum albumin. When oleate or palmitate was added in amounts up to 2:1 excess to human serum albumin solutions the measured rate constant for the transition (k2) was increased in comparison with fatty acid free albumin. When the fatty acid concentration is further increased, k2 decreases. When the fatty acid level is 2 to 3 mol per mol albumin, the affinity constant (KA) is higher than for fatty acid free solutions. With higher ratios the value for KA is reduced. According to the observed changes in kinetic parameters, the binding of warfarin is apparently affected allosterically. A reduced plasma protein binding of coumarins should be expected when fatty acid levels are raised over a prolonged period.

[Release, absorption and elimination of theophylline from fast and slowly released oral formulations]. / Untersuchungen zur Liberation, Absorption und Elimination von Theophyllin bei rasch und verzögert freisetzenden oralen Arzneiformen.

Each 2 tablets of four tablet formulations with 150 mg theophylline were administered to 6 and 5 volunteers, respectively, as single oral dose. 8 volunteers received 256 mg theophylline as a solution and as a sustained released formulation, as well as 176 mg theophylline as short intravenous bolus infusion. The elimination was independent of the examined formulations, but differences occurred between the experiments with the different groups of volunteers. The invasion parameters (t1/2i) of the four fast released tablet formulations corresponded to the values (t1/2a) of the oral theophylline solution. Furthermore, no difference existed concerning the mean times (Tsys). The mean time (theophylline) for the body model, Tvss, is 9.9 h; the mean time, which is attributed to the absorption process (Tabs) is 0.7 h; the mean in vivo dissolution time (Tdiss-vivo) for the sustained release formulation is 6.3 h. The mean time after oral administration of the theophylline solution (Tbiol) is 10.6 h. General conditions for a comparison between the in vitro and the in vivo release data are reported.

Stopped-flow studies on drug-protein binding. Analog-computer analysis of the pH-dependent binding kinetics of warfarin and human serum albumin.

Binding curves obtained by the stopped-flow method for the association of warfarin and human serum albumin (HSA) at pH 6.0 and 9.0 have been analysed with digital- and analog computers. The first association product (WHSA') at pH 6-9 does not contribute to the observed fluorescence enhancement during warfarin-HSA complex formation. A similar consecutive relaxation process leads to a more stable warfarin-HSA complex, with HSA in the neutral (N)-form (pH 6) and base (B)-form (pH 9). This rearrangement can be measured by stopped-flow (k 2 = 31 s-1 at pH 6; K'2 = 63 s-1 at pH 9). At pH 6 a further concentration dependent relaxation process has been observed indicating that the complex of warfarin with the N-form of HSA gets partially converted into its B-form with a half-time for this N leads to B transition in the range of 0.2-0.4 s. A drug such as warfarin can act as effector molecule for conformational changes of the HSA tertiary and quaternary structure during the formation of a high affinity complex.

Stopped-flow studies on drug-protein binding. 1. Kinetics of warfarin binding to human serum albumin.

We have studied the binding of warfarin to human serum albumin (HSA) with the stopped-flow method. At 37 degrees C the rate constant for the velocity of dissociation of the stable warfarin-HSA complex is 10 S-1 (t50% = 0.07 s). Concentration and temperature dependent association constants for warfarin binding to HSA have been measured (2.5 x 10(5) M-1 S-1 at 6 degrees C, 9.8 x 10(5) M-1 S-1 at 22 degrees C and 15.3 x 10(5) M-1 S-1 at 37 degrees C). Our experimentally obtained relaxation constants are best explained by the existence of 5 equivalent low affinity binding sites for warfarin on the HSA molecule, each capable of conversion into a high affinity site. The measured energy of activation for this conversion is 57.5 kJ M-1.