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.

Low energy expenditure and resting behaviour of humpback whale mother-calf pairs highlights conservation importance of sheltered breeding areas.

Understanding the behaviour of humpback whale mother-calf pairs and the acoustic environment on their breeding grounds is fundamental to assessing the biological and ecological requirements needed to ensure a successful migration and survival of calves. Therefore, on a breeding/resting ground, Exmouth Gulf, Western Australia, we used animal-borne DTAGs to quantify the fine-scale behaviour and energetic expenditure of humpback whale mothers and calves, while sound recorders measured the acoustic environment. We show that: (i) lactating humpback whales keep their energy expenditure low by devoting a significant amount of time to rest, and their use of energy, inferred from respiration rates, is ~half than that of adults on their foraging grounds; (ii) lactating females mainly rest while stationary at shallow depths within reach of the hull of commercial ships, thus increasing the potential for ship strike collisions; (iii) the soundscape is dominated by biological sources; and (iv) even moderate increases of noise from vessels will decrease the communication range of humpback whales. Planned commercial infrastructure in Exmouth Gulf will cause a substantial increase in shipping traffic with the risk of ship strikes and acoustic disturbance potentially compromising energy reserves for the southern migration of humpback whales.

Inhibition of 3-hydroxy-3-methylglutaryl-CoA synthase and cholesterol biosynthesis by beta-lactone inhibitors and binding of these inhibitors to the enzyme.

The beta-lactones L-659,699 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2- oxetanyl]-3,5,7-trimethyl-2,4-undecadienoic acid) and its radioactive derivative 3H-L-668,411 (the 2,3-ditritiated methyl ester of L-659,699) inhibited a partially purified preparation of rat liver cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase with an IC50 of 0.1 microM. These compounds were also found to inhibit the incorporation of [14C]acetate into sterols in cultured Hep G2 cells with an IC50 of 3 microM. New kinetic evidence indicated that inhibition of the isolated enzyme was irreversible. In contrast, sterol biosynthesis in cultured Hep G2 cells was rapidly restored upon removal of the compound from the medium of inhibited cultures, suggesting reversibility of inhibition in the cells. Radioactivity was found to be associated with a single cytoplasmic protein by SDS/PAGE of the cytoplasm of Hep G2 cells after incubation of the cells with the inhibitor 3H-L-668,411. This protein was identified as cytoplasmic HMG-CoA synthase. Binding of the radioactive compound to the enzyme was decreased with time if the radioactive inhibitor was removed from the medium. Exposure of a gel containing the radioactive enzyme-inhibitor complex to neutral hydroxylamine also resulted in a loss of radioactivity from the gel. The purified rat liver enzyme reacted with the 3H-ligand to form a stable enzyme-inhibitor complex which could be isolated by h.p.l.c. Radioactivity was also subsequently lost from this complex when it was incubated with neutral hydroxylamine. Incorporation of [14C]acetate into cholesterol in mouse liver was inhibited in a reversible manner after oral administration of the beta-lactone inhibitor. These studies, as well as the kinetic evidence presented, suggest that the beta-lactone inhibitors acylate HMG-CoA synthase in a reaction which appears to be irreversible in vitro, but is easily reversed in cultured cells and in animals.

The inhibition of cytoplasmic acetoacetyl-CoA thiolase by a triyne carbonate (L-660, 631).

The compound L-660, 631 (2-oxo-5-(1-hydroxy-2,4,6-heptatriynyl)-1,3-dioxolane-4 heptanoic acid), a natural product isolated from an Actinomycete culture, was found to inhibit rat liver cytosolic acetoacetyl-CoA thiolase, the first step in the cholesterol biosynthesis pathway, with an IC50 of 1.0 x 10(-8) M. The inhibitor had no effect on other sulfhydryl containing enzymes of lipid synthesis such as HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. When tested in cultured human liver Hep G2 cells the compound inhibited the incorporation of 14C-acetate and 14C-octanoate into sterols 56% and 48% respectively at 3 x 10(-6) M with no effect on fatty acid synthesis. No noticeable effect was seen on fatty acid biosynthesis. This strongly suggests that the locus of inhibition of acetate incorporation into sterols found with this compound is the acetoacetyl-CoA thiolase step in the cholesterol biosynthesis pathway.

Separation and identification of triglycerides, cholesteryl esters, cholesterol, 7-dehydrocholesterol, dolichol, ubiquinone, alpha-tocopherol, and retinol by high performance liquid chromatography with a diode array detector.

A simple isocratic high performance liquid chromatography (HPLC) system is described that allows separation and identification of cholesteryl esters, triglycerides, ubiquinone, alpha-tocopherol, dolichol, cholesterol, 7-dehydrocholesterol, and retinol. This consisted of a normal phase cyanopropyl column with 0.1% isopropanol in heptane as the solvent. The effluent was monitored with an LKB model 2140 diode array detector which enabled the lipids to be identified by their characteristic absorption spectra. This system was applied to a sample of dog liver in which cholesteryl esters, retinyl esters, triglycerides, ubiquinone, dolichol, cholesterol, and retinol were identified. Retinyl esters and vitamin D esters were identified by their similarity in absorption spectra to retinol and vitamin D. A system to transfer and store the chromatograms on the VAX PDP-11 or an optical disc is also described.