We discuss the current status of liquid biopsy and its advantages and challenges with a focus on pre-analytical sample handling, technologies and workflows. The potential of circulating tumor cells ...and circulating tumor DNA is pointed out and an overview of corresponding technologies is given.
In human body fluids, microRNA (miRNA) can be found as circulating cell-free miRNA (cfmiRNA), as well as secreted into extracellular vesicles (EVmiRNA). miRNAs are being intensively evaluated as ...minimally invasive liquid biopsy biomarkers in patients with cancer. The growing interest in developing clinical assays for circulating miRNA necessitates careful consideration of confounding effects of preanalytical and analytical parameters.
By using reverse transcription quantitative real-time PCR and next-generation sequencing (NGS), we compared extraction efficiencies of 5 different protocols for cfmiRNA and 2 protocols for EVmiRNA isolation in a multicentric manner. The efficiency of the different extraction methods was evaluated by measuring exogenously spiked cel-miR-39 and 6 targeted miRNAs in plasma from 20 healthy individuals.
There were significant differences between the tested methods. Although column-based extraction methods were highly effective for the isolation of endogenous miRNA, phenol extraction combined with column-based miRNA purification and ultracentrifugation resulted in lower quality and quantity of isolated miRNA. Among all extraction methods, the ubiquitously expressed miR-16 was represented with high abundance when compared with other targeted miRNAs. In addition, the use of miR-16 as an endogenous control for normalization of quantification cycle values resulted in a decreased variability of column-based cfmiRNA extraction methods. Cluster analysis of normalized NGS counts clearly indicated a method-dependent bias.
The choice of plasma miRNA extraction methods affects the selection of potential miRNA marker candidates and mechanistic interpretation of results, which should be done with caution, particularly across studies using different protocols.
In this paper we first of all summarize and rationalize current typologies of organizational forms, arranging available classifications in a hierarchy of increasing generality. The ensuing structure ...parallels the classification of living beings into classes of increasing generality such as species, genus, family, order, and so on. Subsequently, we analyze the structure of communications that favored the diffusion of each organizational form. We isolate a few stylized communication structures, pointing to the presence of several sources endowed with global connections as the most efficient diffusion mode. The empirical research that is being carried out on single organizations is close to observing their T-patterns, whereas nothing comparable is in sight for organizational forms as yet. However, at least in some cases, we dare to formulate tentative hypotheses on certain features that the ensuing T-patterns-of-patterns might exhibit.
This article investigates the potential of selected urban typologies in Vienna to reach the state of Positive Energy Districts (PED) by achieving a positive annual energy balance. It follows the EU ...initiative for implementing at least 100 PED in Europe by 2025. Four urban typologies have been assessed using the bottom-up energy modelling tool MAPED that enables a simplified energy demand-supply analysis at the district scale. Considering relevant urban typologies in different construction periods, the analysis focused on converting the allocated building stocks into PED by employing comprehensive thermal refurbishment and energy efficiency measures, electrification of end-uses and fuel switching, exploitation of local renewable energy potential, and flexible interaction with the regional energy system. The results reveal that a detached housing district can achieve a positive annual energy balance (for heat and power) of 110% due to the fact that there are sufficient surfaces (roofs, facades, open land) available for the production of local renewable energy, whereas the remaining typologies fail to achieve the criteria with an annual balance ranking between 61% and 97%, showing additional margins for improvement to meet the PED conditions. The presented concept offers a practical approach to investigate the PED suitability of urban typologies. It will help the Austrian Ministry for Climate Action and Environment to identify appropriate strategies for the refurbishment of existing urban areas towards the PED standard.
At a global level, the need for energy efficiency and an increased share of renewable energy sources is evident, as is the crucial role of cities due to the rapid urbanization rate. As a consequence ...of this, the research work related to Positive Energy Districts (PED) has accelerated in recent years. A common shared definition, as well as technological approaches or methodological issues related to PEDs are still unclear in this development and a global scientific discussion is needed. The International Energy Agency’s Energy in Buildings and Communities Programme (IEA EBC) Annex 83 is the main platform for this international scientific debate and research. This paper describes the challenges of PEDs and the issues that are open for discussions and how the Annex 83 is planned and organized to facilitate this and to actively steer the development of PEDs major leaps forward. The main topics of discussion in the PED context are the role and importance of definitions of PEDs, virtual and geographical boundaries in PEDs, the role of different stakeholders, evaluation approaches, and the learnings of realized PED projects.
Philadelphia-like B-cell precursor acute lymphoblastic leukemia (Ph-like ALL) is characterized by distinct genetic alterations and inferior prognosis in children and younger adults. The purpose of ...this study was a genetic and clinical characterization of Ph-like ALL in adults. Twenty-six (13%) of 207 adult patients (median age: 42 years) with B-cell precursor ALL (BCP-ALL) were classified as having Ph-like ALL using gene expression profiling. The frequency of Ph-like ALL was 27% among 95 BCP-ALL patients negative for BCR-ABL1 and KMT2A-rearrangements. IGH-CRLF2 rearrangements (6/16; P=0.002) and mutations in JAK2 (7/16; P<0.001) were found exclusively in the Ph-like ALL subgroup. Clinical and outcome analyses were restricted to patients treated in German Multicenter Study Group for Adult ALL (GMALL) trials 06/99 and 07/03 (n=107). The complete remission rate was 100% among both Ph-like ALL patients (n=19) and the "remaining BCP-ALL" cases (n=40), i.e. patients negative for BCR-ABL1 and KMT2A-rearrangements and the Ph-like subtype. Significantly fewer Ph-like ALL patients reached molecular complete remission (33% versus 79%; P=0.02) and had a lower probability of continuous complete remission (26% versus 60%; P=0.03) and overall survival (22% versus 64%; P=0.006) at 5 years compared to the remaining BCP-ALL patients. The profile of genetic lesions in adults with Ph-like ALL, including older adults, resembles that of pediatric Ph-like ALL and differs from the profile in the remaining BCP-ALL. Our study is the first to demonstrate that Ph-like ALL is associated with inferior outcomes in intensively treated older adult patients. Ph-like adult ALL should be recognized as a distinct, high-risk entity and further research on improved diagnostic and therapeutic approaches is needed. (NCT00199056, NCT00198991).
Circulating tumor cells (CTCs), potential precursors of most epithelial solid tumors, are mainly enriched by epithelial cell adhesion molecule (EpCAM)-dependent technologies. Hence, these approaches ...may overlook mesenchymal CTCs, considered highly malignant. Our aim was to establish a workflow to enrich and isolate patient-matched EpCAMhigh and EpCAMlow/negative CTCs within the same blood samples, and to investigate the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutational status within single CTCs. We sequentially processed metastatic breast cancer (MBC) blood samples via CellSearch® (EpCAM-based) and via Parsortix™ (size-based) systems. After enrichment, cells captured in Parsortix™ cassettes were stained in situ for nuclei, cytokeratins, EpCAM and CD45. Afterwards, sorted cells were isolated via CellCelector™ micromanipulator and their genomes were amplified. Lastly, PIK3CA mutational status was analyzed by combining an amplicon-based approach with Sanger sequencing. In 54% of patients′ blood samples both EpCAMhigh and EpCAMlow/negative cells were identified and successfully isolated. High genomic integrity was observed in 8% of amplified genomes of EpCAMlow/negative cells vs. 28% of EpCAMhigh cells suggesting an increased apoptosis in the first CTC-subpopulation. Furthermore, PIK3CA hotspot mutations were detected in both EpCAMhigh and EpCAMlow/negative CTCs. Our workflow is suitable for single CTC analysis, permitting—for the first time—assessment of the heterogeneity of PIK3CA mutational status within patient-matched EpCAMhigh and EpCAMlow/negative CTCs.
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