High-throughput molecular assays for inclusion in personalised oncology trials – State-of-the-art and beyond

In the last decades, the development of high-throughput molecular assays has revolutionised cancer diagnostics, paving the way for the concept of personalised cancer medicine. This progress has been driven by the introduction of such technologies through biomarker-driven oncology trials. In this review, strengths and limitations of various state-of-the-art sequencing technologies, including gene panel sequencing (DNA and RNA), whole-exome/whole-genome sequencing and whole-transcriptome sequencing, are explored, focusing on their ability to identify clinically relevant biomarkers with diagnostic, prognostic and/or predictive impact. This includes the need to assess complex biomarkers, for example microsatellite instability, tumour mutation burden and homologous recombination deficiency, to identify patients suitable for specific therapies, including immunotherapy. Furthermore, the crucial role of biomarker analysis and multidisciplinary molecular tumour boards in selecting patients for trial inclusion is discussed in relation to various trial concepts, including drug repurposing. Recognising that today's exploratory techniques will evolve into tomorrow's routine diagnostics and clinical study inclusion assays, the importance of emerging technologies for multimodal diagnostics, such as proteomics and in vivo drug sensitivity testing, is also discussed. In addition, key regulatory aspects and the importance of patient engagement in all phases of a clinical trial are described. Finally, we propose a set of recommendations for consideration when planning a new precision cancer medicine trial.imag

High-throughput molecular assays for inclusion in personalised oncology trials – State-of-the-art and beyond

In the last decades, the development of high-throughput molecular assays has revolutionised cancer diagnostics, paving the way for the concept of personalised cancer medicine. This progress has been driven by the introduction of such technologies through biomarker-driven oncology trials. In this review, strengths and limitations of various state-of-the-art sequencing technologies, including gene panel sequencing (DNA and RNA), whole-exome/whole-genome sequencing and whole-transcriptome sequencing, are explored, focusing on their ability to identify clinically relevant biomarkers with diagnostic, prognostic and/or predictive impact. This includes the need to assess complex biomarkers, for example microsatellite instability, tumour mutation burden and homologous recombination deficiency, to identify patients suitable for specific therapies, including immunotherapy. Furthermore, the crucial role of biomarker analysis and multidisciplinary molecular tumour boards in selecting patients for trial inclusion is discussed in relation to various trial concepts, including drug repurposing. Recognising that today's exploratory techniques will evolve into tomorrow's routine diagnostics and clinical study inclusion assays, the importance of emerging technologies for multimodal diagnostics, such as proteomics and in vivo drug sensitivity testing, is also discussed. In addition, key regulatory aspects and the importance of patient engagement in all phases of a clinical trial are described. Finally, we propose a set of recommendations for consideration when planning a new precision cancer medicine trial.imag

Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O.

Niemann-Pick disease type C (NPC) is caused by defects in cholesterol efflux from lysosomes due to mutations of genes coding for NPC1 and NPC2 proteins. As a result, massive accumulation of unesterified cholesterol in late endosomes/lysosomes is observed. At the level of the organism these cholesterol metabolism disorders are manifested by progressive neurodegeneration and hepatosplenomegaly. Until now filipin staining of cholesterol deposits in cells has been widely used for NPC diagnostics. In this report we present an alternative method for cholesterol visualization and estimation using a cholesterol-binding bacterial toxin, perfringolysin O

A 20-year longitudinal study of plasma chitotriosidase activity in treated Gaucher disease type 1 and 3 patients-a qualitative and quantitative approach

Chitotriosidase is an enzyme produced and secreted in large amounts by activated macrophages, especially macrophages loaded with phagocytozed glycosphingolipid in Gaucher disease. Macrophages phagocytose decayed blood cells that contain a lot of sphingolipid-rich cell membranes. In Gaucher disease, due to a deficit in beta-glucocerebrosidase activity, the phagocytozed substrate glucocerebroside cannot undergo further catabolism. In such a situation, macrophages secrete chitotriosidase in proportion to the degree of overload. Gaucher disease (GD) is a recessively inherited disorder resulting in storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. It is directly caused by the deficiency of beta-glucocerebrosidase (GBA) activity. Chitotriosidase has been measured systematically each year in the same group of 49 patients with type 1 and 3 GD for over 20 years. Our analysis showed that chitotriosidase is very sensitive biomarker to enzyme replacement therapy (ERT). The response to treatment introduction is of an almost immediate nature, lowering pathologically high chitotriosidase levels by a factor of 2 in a time scale of 8 months, on average. Long term enzyme replacement therapy (ERT) brings chitotriosidase activity close to reference values. Finally, reducing the dose of ERT quickly boosts chitotriosidase activity, but restoring the initial dose of treatment brings chitotriosidase level of activity back onto the decreasing time trajectory

Expanding the genetic cause of multiple sulfatase deficiency: A novel SUMF1 variant in a patient displaying a severe late infantile form of the disease

Jaszczuk I, Schlotawa L, Dierks T, et al. Expanding the genetic cause of multiple sulfatase deficiency: A novel SUMF1 variant in a patient displaying a severe late infantile form of the disease. MOLECULAR GENETICS AND METABOLISM. 2017;121(3):252-258.Multiple sulfatase deficiency (MSD) is a rare inherited metabolic disease caused by defective cellular sulfatases. Activity of sulfatases depends on post-translational modification catalyzed by formylglycine-generating enzyme (FGE), encoded by the SUMF1 gene. SUMF1 pathologic variants cause MSD, a syndrome presenting with a complex phenotype. We describe the first Polish patient with MSD caused by a yet undescribed pathologic variant c.337G>A [p.Glu113Lys] (i.e. p.E113K) in heterozygous combination with the known deletion allele c.519 +5_519 + 8del [p.Ala149_Ala173del]. The clinical picture of the patient initially suggested late infantile metachromatic leukodystrophy, with developmental delay followed by regression of visual, hearing and motor abilities as the most apparent clinical symptoms. Transient signs of ichthyosis and minor dysmorphic features guided the laboratory workup towards MSD. Since MSD is a rare disease and there is a variable clinical spectrum, we thoroughly describe the clinical outcome of our patient. The FGE-E113K variant, expressed in cell culture, correctly localized to the endoplasmic reticulum but was retained intracellularly in contrast to the wild type FGE. Analysis of FGE-mediated activation of steroid sulfatase in immortalized MSD cells revealed that FGE-E113K exhibited only approx. 15% of the activity of wild type FGE. Based on the crystal structure we predict that the exchange of glutamate-113 against lysine should induce a strong destabilization of the secondary structure, possibly affecting the folding for correct disulfide bridging between C235-C346 as well as distortion of the active site groove that could affect both the intracellular stability as well as the activity of FGE. Thus, the novel variant of the SUMF1 gene obviously results in functionally impaired FGE protein leading to a severe late infantile type of MSD. (C) 2017 Elsevier Inc. All rights reserved