Detection of tumor ALK status in neuroblastoma patients using peripheral blood

International audienceNew protocols based on ALK-targeted therapy by crizotinib or other ALK-targeting molecules have opened for the treatment of patients with neuroblastoma (NB) if their tumors showed mutation and/or amplification of the ALK gene. However, tumor samples are not always available for analysis of ALK mutational status in particular at relapse. Here, we evaluated the ALK mutational status of NB samples by analysis of circulating DNA, using the droplet digital PCR (ddPCR) system. ddPCR assays was developed for the detection of ALK mutations at F1174 and R1275 hotspots found in NB tumors and was applied for the analysis of circulating DNA obtained from 200L of serum or plasma samples collected from 114 patients with NB. The mutations F1174L (exon 23 position 3520, T>C and position 3522, C>A) and the mutation R1275Q (exon 25 position 3824, G>A) were detected in circulating DNA. The sensitivity of our test was 100%, 85%, and 92%, respectively, and the specificity was 100%, 91%, and 98%, respectively. In conclusion, the assay that we have developed offers a reliable, noninvasive blood test to assess ALK mutational status at F1174 and R1275 hotspots and should help clinicians to identify patients showing an ALK mutation in particular when no tumor tissue is available

How does conformational flexibility influence key structural features involved in activation of anaplastic lymphoma kinase?

Anaplastic Lymphoma Kinase (ALK) plays a major role in developing tumor processes and therefore has emerged as a validated therapeutic target. Applying atomistic molecular dynamics simulations on the wild type enzyme and the nine most frequently occurring and clinically important activation mutants we revealed important conformational effects on key interactions responsible for the activation of the enzyme

Empty rituals? A qualitative study of users’ experience of monitoring & evaluation systems in HIV interventions in western India

In global health initiatives, particularly in the context of private philanthropy and its ‘business minded’ approach, detailed programme data plays an increasing role in informing assessments, improvements, evaluations, and ultimately continuation or discontinuation of funds for individual programmes. The HIV/AIDS literature predominantly treats monitoring as unproblematic. However, the social science of audit and indicators emphasises the constitutive power of indicators, noting that their effects at a grassroots level are often at odds with the goals specified in policy. This paper investigates users' experiences of Monitoring and Evaluation (M&E) systems in the context of HIV interventions in western India. Six focus groups (totalling 51 participants) were held with employees of 6 different NGOs working for government or philanthropy-funded HIV interventions for sex workers in western India. Ten donor employees were interviewed. Thematic analysis was conducted. NGO employees described a major gap between what they considered their “real work” and the indicators used to monitor it. They could explain the official purposes of M&E systems in terms of programme improvement and financial accountability. More cynically, they valued M&E experience on their CVs and the rhetorical role of data in demonstrating their achievements. They believed that inappropriate and unethical means were being used to meet targets, including incentives and coercion, and criticised indicators for being misleading and inflexible. Donor employees valued the role of M&E in programme improvement, financial accountability, and professionalising NGO-donor relationships. However, they were suspicious that NGOs might be falsifying data, criticised the insensitivity of indicators, and complained that data were under-used. For its users, M& E appears an ‘empty ritual’, enacted because donors require it, but not put to local use. In this context, monitoring is constituted as an instrument of performance management rather than as a means of rational programme improvement

Seamless Integration of Heterogeneous Devices and Access Control in Smart Homes

Abstract—The recent trend of ubiquitous access to embedded physical devices over the Internet as well as increasing penetration of wireless protocols such as ZigBee has raised attention to smart homes. These systems consist of sensors, devices and smart appliances that can be monitored and controlled remotely by human users and cloud services. However, the lack of a de facto communication standard for smart homes creates a barrier against the interoperability of devices from different vendors. We address this challenge by proposing a holistic, extensible software architecture that seamlessly integrates heterogeneous protocol- and vendor-specific devices and services, while making these services securely available over the Internet. Our architecture is developed on top of the OSGi framework and incorporates a semantic model of a smart home system. As a result, we achieve semantic interoperability – the ability to integrate new applications and drivers into the deployed system during runtime. Furthermore, we integrate a new access control model for specific smart home scenarios. As a proof of our concept, we demonstrate the seamless semantic discovery of home devices at runtime by integrating several protocols including X10, Insteon, ZigBee and UPnP into a real test. Using smart phones and cloud services together with our home gateway implementation, we further demonstrate the ease of integration of new applications and drivers. Keywords- smart home; interoperation; semantics; access control I

Timing analysis for embedded systems using non-preemptive EDF scheduling under bounded error arrivals

Embedded systems consist of one or more processing units which are completely encapsulated by the devices under their control, and they often have stringent timing constraints associated with their functional specification. Previous research has considered the performance of different types of task scheduling algorithm and developed associated timing analysis techniques for such systems. Although preemptive scheduling techniques have traditionally been favored, rapid increases in processor speeds combined with improved insights into the behavior of non-preemptive scheduling techniques have seen an increased interest in their use for real-time applications such as multimedia, automation and control. However when non-preemptive scheduling techniques are employed there is a potential lack of error confinement should any timing errors occur in individual software tasks. In this paper, the focus is upon adding fault tolerance in systems using non-preemptive deadline-driven scheduling. Schedulability conditions are derived for fault-tolerant periodic and sporadic task sets experiencing bounded error arrivals under non-preemptive deadline scheduling. A timing analysis algorithm is presented based upon these conditions and its run-time properties are studied. Computational experiments show it to be highly efficient in terms of run-time complexity and competitive ratio when compared to previous approaches

Anaplastic lymphoma kinase (ALK) inhibitor response in neuroblastoma is highly correlated with ALK mutation status, ALK mRNA and protein levels

Background In pediatric neuroblastoma (NBL), high anaplastic lymphoma kinase (ALK) levels appear to be correlated with an unfavorable prognosis, regardless of ALK mutation status. This suggests a therapeutic role for ALK inhibitors in NBL patients. We examined the correlation between levels of ALK, phosphorylated ALK (pALK) and downstream signaling proteins and response to ALK inhibition in a large panel of both ALK mutated and wild type (WT) NBL cell lines. Methods We measured protein levels by western blot and ALK inhibitor sensitivity (TAE684) by viability assays in 19 NBL cell lines of which 6 had a point mutation and 4 an amplification of the ALK gene. Results ALK 220 kDa (p=0.01) and ALK 140 kDa (p= 0.03) protein levels were higher in ALK mutant than WT cell lines. Response to ALK inhibition was significantly correlated with ALK protein levels (p<0.01). ALK mutant cell lines (n=4) were 14,9 fold (p<0,01) more sensitive to ALK inhibition than eight WT cell lines. Conclusion NBL cell lines often express ALK at high levels and are responsive to ALK inhibitors. Mutated cell lines express ALK at higher levels, which may define their superior response to ALK inhibition

In search of the authentic nation: landscape and national identity in Canada and Switzerland

While the study of nationalism and national identity has flourished in the last decade, little attention has been devoted to the conditions under which natural environments acquire significance in definitions of nationhood. This article examines the identity-forming role of landscape depictions in two polyethnic nation-states: Canada and Switzerland. Two types of geographical national identity are identified. The first – what we call the ‘nationalisation of nature’– portrays zarticular landscapes as expressions of national authenticity. The second pattern – what we refer to as the ‘naturalisation of the nation’– rests upon a notion of geographical determinism that depicts specific landscapes as forces capable of determining national identity. The authors offer two reasons why the second pattern came to prevail in the cases under consideration: (1) the affinity between wild landscape and the Romantic ideal of pure, rugged nature, and (2) a divergence between the nationalist ideal of ethnic homogeneity and the polyethnic composition of the two societies under consideration

Low aerobic mitochondrial energy metabolism in poorly- or undifferentiated neuroblastoma

<p>Abstract</p> <p>Background</p> <p>Succinate dehydrogenase (SDH) has been associated with carcinogenesis in pheochromocytoma and paraganglioma. In the present study we investigated components of the oxidative phosphorylation system in human neuroblastoma tissue samples.</p> <p>Methods</p> <p>Spectrophotometric measurements, immunohistochemical analysis and Western blot analysis were used to characterize the aerobic mitochondrial energy metabolism in neuroblastomas (NB).</p> <p>Results</p> <p>Compared to mitochondrial citrate synthase, SDH activity was severely reduced in NB (n = 14) versus kidney tissue. However no pathogenic mutations could be identified in any of the four subunits of SDH. Furthermore, no genetic alterations could be identified in the two novel SDH assembly factors SDHAF1 and SDH5. Alterations in genes encoding nfs-1, frataxin and isd-11 that could lead to a diminished SDH activity have not been detected in NB.</p> <p>Conclusion</p> <p>Because downregulation of other complexes of the oxidative phosphorylation system was also observed, a more generalized reduction of mitochondrial respiration seems to be present in neuroblastoma in contrast to the single enzyme defect found in hereditary pheochromocytomas.</p

Verification of genes differentially expressed in neuroblastoma tumours: a study of potential tumour suppressor genes

<p>Abstract</p> <p>Background</p> <p>One of the most striking features of the childhood malignancy neuroblastoma (NB) is its clinical heterogeneity. Although there is a great need for better clinical and biological markers to distinguish between tumours with different severity and to improve treatment, no clear-cut prognostic factors have been found. Also, no major NB tumour suppressor genes have been identified.</p> <p>Methods</p> <p>In this study we performed expression analysis by quantitative real-time PCR (QPCR) on primary NB tumours divided into two groups, of favourable and unfavourable outcome respectively. Candidate genes were selected on basis of lower expression in unfavourable tumour types compared to favourables in our microarray expression analysis. Selected genes were studied in two steps: (1) using TaqMan Low Density Arrays (TLDA) targeting 89 genes on a set of 12 NB tumour samples, and (2) 12 genes were selected from the TLDA analysis for verification using individual TaqMan assays in a new set of 13 NB tumour samples.</p> <p>Results</p> <p>By TLDA analysis, 81 out of 87 genes were found to be significantly differentially expressed between groups, of which 14 have previously been reported as having an altered gene expression in NB. In the second verification round, seven out of 12 transcripts showed significantly lower expression in unfavourable NB tumours, <it>ATBF1</it>, <it>CACNA2D3</it>, <it>CNTNAP2</it>, <it>FUSIP1</it>, <it>GNB1</it>, <it>SLC35E2</it>, and <it>TFAP2B</it>. The gene that showed the highest fold change in the TLDA analysis, <it>POU4F2</it>, was investigated for epigenetic changes (CpG methylation) and mutations in order to explore the cause of the differential expression. Moreover, the fragile site gene <it>CNTNAP2 </it>that showed the largest fold change in verification group 2 was investigated for structural aberrations by copy number analysis. However, the analyses of <it>POU4F2 </it>and <it>CNTNAP2 </it>showed no genetic alterations that could explain a lower expression in unfavourable NB tumours.</p> <p>Conclusion</p> <p>Through two steps of verification, seven transcripts were found to significantly discriminate between favourable and unfavourable NB tumours. Four of the transcripts, <it>CACNA2D3</it>, <it>GNB1</it>, <it>SLC35E2</it>, and <it>TFAP2B</it>, have been observed in previous microarray studies, and are in this study independently verified. Our results suggest these transcripts to be markers of malignancy, which could have a potential usefulness in the clinic.</p