Development of novel methodological approaches to detection and quantitative monitoring of point-mutated clones

Bei Patienten mit chronisch myeloischer Leukämie (CML) ist das Auftreten von Punktmutationen in der BCR-ABL1 Tyrosinkinasedomäne (TK) die am häufigsten beschriebene Ursache für Resistenzen gegen Tyrosinkinase-Inhibitoren (TKI). Für die Detektion von Punktmutationen ist eine Auswahl an Methoden verfügbar. Diese sind jedoch oft wenig sensitiv, wie zum Beispiel die Standardmethode der bidirektionalen Sequenzierung, mit einer Sensitivität von circa 20%. Das Auftreten von TKI resistenten Zellklonen muss jedoch nicht notwendigerweise mit einer bevorstehenden Progression der Krankheit assoziiert sein. Die Analyse von mutanten Zellklonen mit qualitativen Methoden könnte daher unzureichend sein, um klinisch relevante Klone zu identifizieren. Um diese Problematik bearbeiten zu können, wäre eine Methode von Vorteil, die die Detektion von Punktmutationen in einem klinisch relevanten Bereich mit der Möglichkeit zur Quantifizierung vereint. Solch eine Methode galt es zu entwickeln. Die neu etablierte LD-PCR Methode basiert auf der Hybridisierung spezifischer Sonden an die Wildtyp (WT) bzw. mutierte Sequenz gefolgt von einer ligations-abhängigen kompetitiven PCR. Die generierten Amplikons werden mittels Kapillarelektrophorese detektiert und quantifiziert. LD-PCR Assays wurden für folgende 21 häufig vorkommenden BCR-ABL1 TK Punktmutationen etabliert: M244V, L248V, V299L-C/T, G250E, Q252H-C/T, Y253F, Y253H, E255K, T315A, T315I, F317C, F317I, F317L-A/G, F317V, M351T, F359V, H396P and H396R. Das Konzept der LD-PCR ist auf jegliche Punktmutation umlegbar, wie am Beispiel der Punktmutation V617V im JAK2 Gen, die bei myeloproliferativen Erkrankungen prognostisch bedeutend ist, gezeigt werden konnte. Die LD-PCR Assays haben eine Sensitivität von 1-5% und ermöglichen eine quantitative Überwachung während des Behandlungsverlaufs. Die Anwendbarkeit dieser Methode bei CML Patienten unter TKI Behandlung konnte ebenso gezeigt werden wie das Vorhandensein einer Proliferationskinetik mutierter Zellklone. Die Dokumentation der Größe und der Expansion eines mutierten Zellklons könnte die Vorhersage einer bevorstehenden Resistenz ermöglichen und bei der klinischen Überwachung sowie bei der Therapiesteuerung von CML Patienten hilfreich sein.In patients with chronic myeloid leukemia (CML), the occurrence of point mutations within the BCR-ABL1 tyrosine kinase (TK) domain is currently the most common mechanism of resistance to therapy with TK inhibitors. To date a variety of different techniques to the detection of relevant point mutations is available. This mainly includes methods displaying a limited level of sensitivity e.g. bidirectional sequencing, the standard method of choice, providing a sensitivity of ~ 20%. However, the presence of cells carrying resistant mutations does not necessarily imply imminent disease progression. Mutation analysis by qualitative methods may therefore be insufficient to reliably identify clinically relevant resistant clones. To address this problem, an application permitting the detection of point mutated subclones of clinically relevant size and the additional option for quantitative analysis might be of great benefit. Thus the aim was to establish a technique combining both requirements. The newly developed LD-PCR relies on specific probe hybridization to mutant and wild-type sequences followed by ligation-dependent competitive PCR. Amplicons are detected and quantified via fluorescence-based capillary electrophoresis. Assays have been established for 21 common BCR-ABL1 TK point mutations including M244V, L248V, V299L-C/T, G250E, Q252H-C/T, Y253F, Y253H, E255K, T315A, T315I, F317C, F317I, F317L-A/G, F317V, M351T, F359V, H396P and H396R. Moreover the technique is adaptive to any kind of point mutation as shown for the detection of V617F point mutation within the JAK2 gene, relevant for several myeloproliferative disorders. The LD-PCR assays display a detection limit of 1-5% and permit quantitative monitoring of mutant clones during the course of treatment. The applicability in CML patients undergoing treatment with TK inhibitors and the existence of kinetic proliferation of mutated cells clones could be demonstrated. Assessment of the size and proliferation kinetics of clones carrying specific mutations could therefore be instrumental in the clinical surveillance and therapeutic management of CML patients

Low‐Temperature Heat Capacities and Thermodynamic Functions of Some Palladium and Platinum Group Chalcogenides. II. Dichalcogenides; PtS2, PtTe2, and PdTe2

Heat capacities of platinum disulfide, platinum ditelluride, and palladium ditelluride were measured in the range 5° to 350°K. They show the normal sigmoidal temperature dependence with no evidence of transitions or other anomalies. The derived heat capacity equations were integrated. Values of Cp, S°—S0°, H°—H0°, and —(F°—H0°)/T are tabulated for selected temperatures. At 298.15°K the entropies are 17.85 cal gfw—1 °K—1 for PtS2, 28.92 cal gfw—1 °K—1 for PtTe2 and 30.25 cal gfw—1 °K—1 for PdTe2. Thermodynamic values have been estimated for other dichalcogenides and related chalcogenides of the platinum group metals.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69847/2/JCPSA6-35-5-1670-1.pd

A Participatory Process to Develop a Landslide Warning System: Paradoxes of Responsibility Sharing in a Case Study in Upper Austria

During a participatory process in Gmunden, Austria, the organizational and responsibility-sharing arrangements for a landslide warning system proved to be contested issues. While questions on the warning system technology and the distribution of information, including the alarm for evacuation, could be resolved with the support of experts, controversies arose on the financial and legal responsibilities that ensure long-term and effective monitoring for the protection of the landslide-prone community. This paper examines how responsibilities can be shared among the residents, experts, and public authorities during the design and operation of landslide warning systems. In particular, we discuss the outcome and implications of three stakeholder workshops where participants deliberated on warning-system options that, in turn, were based on a discourse analysis of extensive stakeholder interviews. The results of the case study show that an end-user orientation requires the consideration of stakeholder worldviews, interests, and conflicts. Paradoxically, the public did not fully support their own involvement in the maintenance and control of the warning system, but the authorities promoted shared responsibility. Deliberative planning does not then necessarily lead to responsibility sharing, but it proved effective as a platform for information and for shared ownership in the warning syste

Warning System Options for Landslide Risk: A Case Study in Upper Austria

This paper explores warning system options in the landslide-prone community of Gmunden/Gschliefgraben in Upper Austria. It describes stakeholder perspectives on the technical, social, economic, legal and institutional characteristics of a warning system. The perspectives differ on issues such as responsibility allocation in decisions regarding warnings, technologies used for monitoring and forecasting, costs and financial aspects, open data policies and the role of the residents. Drawing on the theory of plural rationality and based on a desk study and interviews, stakeholder perspectives and discourses on the warning system problem and its solution were elicited. The perspectives formed the basis for the specification of three technical policy options for a warning system in Gschliefgraben: a minimal-cost and cost-effective system; a technical-expert system; and a resident-centered system. The case demonstrates the importance of accounting for a plurality of values and preferences and of giving voice to competing discourses in communities contemplating warning systems or other public good policies. This paper concludes that understanding the different and often conflicting perspectives and technical policy options is the starting point for formulating an agreed compromise for an effective warning system. We describe the compromise solution in an accompanying paper included in this Special Issue

Expression of functional neuronal receptor latrophilin 1 in human acute myeloid leukaemia cells

Acute myeloid leukaemia (AML) is a blood cancer affecting cells of myeloid lineage. It is characterised by rapid growth of malignant leukocytes that accumulate in the bone marrow and suppress normal haematopoiesis. This systemic disease remains a serious medical burden worldwide. Characterisation of protein antigens specifically expressed by malignant cells, but not by healthy leukocytes, is vital for the diagnostics and targeted treatment of AML. Here we report, for the first time, that the neuronal receptor latrophilin-1 is expressed in human monocytic leukaemia cell lines and in primary human AML cells. However, it is absent in healthy leukocytes. Latrophilin-1 is functional in leukaemia cells tested, and its biosynthesis is controlled through the mammalian target of rapamycin (mTOR), a master regulator of myeloid cell translational pathways. Our findings demonstrate that latrophilin-1 could be considered as a novel biomarker of human AML, which offers potential new avenues for AML diagnosis and treatment

Quantitative analysis of mutant subclones in chronic myeloid leukemia : comparison of different methodological approaches

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points