Implementierung eines prädiktiven Biomarkers in den diagnostischen Routinealltag am Beispiel der Anaplastischen Lymphom Kinase (ALK) im nicht-kleinzelligen Lungenkarzinom (NSCLC)

Im aktuellen Zeitalter der zielgerichteten Krebstherapien müssen sich pathologische Institute in regelmäßigen Abständen auf immer wieder neu zu testende prädiktive Biomarker einstellen. Das bedeutet, dass potentielle Testverfahren validiert werden müssen, sowie der Nachweis der entsprechenden diagnostischen Güte im Rahmen von Ringversuchen gezeigt werden muss. Je seltener das zu testende Ereignis bei der entsprechenden Erkrankung vorkommt, umso schwieriger wird die Gesamtsituation. Dies erfordert institutsübergreifende Kooperationen. In der vorliegenden Arbeit konnten wir den Stellenwert der Ringversuche am Beispiel der ALK-Alterationen im NSCLC herausarbeiten. Wir konnten zeigen, dass der diagnostische Goldstandard (FISH) unter gewissen Umständen kritisch betrachtet werden muss. Hinsichtlich der in den Ringversuchen eingesetzten ISH-Verfahren (FISH und CISH), sowie bezüglich der verschiedenen Sonden unterschiedlicher Anbieter zeigten sich keine Vor- oder Nachteile. Die Daten zeigen weiterhin, dass nur bestimmte Antikörper-Klone nach Harmonisierung der Methodik im immunhistologisch diagnostischen Alltag verlässlich eingesetzt werden können. Dieser Ansatz ist nicht nur zeit- und kostensparend, sondern verbessert zusätzlich die diagnostische Sicherheit insbesondere bei den „Borderline“-Fällen. Bisherige und zukünftig NGS-basierte Daten werden dabei helfen, die IHC nicht nur als Screening-Methode, sondern gegebenenfalls als „stand-alone“ Test zu etablieren. Dennoch, in immunhistologisch unklaren Fällen sollte mindestens eine weitere Methode (ISH, NGS) angewendet werden können. Hierbei ist das Wissen um deren Vor- und Nachteile und der damit verbundenen Ergebnisinterpretation entscheidend. In der Folge wird die FISH, welche die Methode der Wahl in den zur Medikamentenzulassung führenden Studien war, möglicherweise an ihrer zentralen Bedeutung verlieren. Begründet ist dies nicht nur in etwaigen technischen Artefakten, auch wirtschaftliche Argumente (Kosten, technischer und zeitlicher Aufwand, Bearbeitungszeit) spielen letztendlich eine Rolle. Ein Screening aller Lungenkrebsfälle mittels FISH ist nicht in jeder Institution umzusetzen, mittels IHC ist dies leichter möglich. Ferner erlauben es die zur Verfügung stehenden NGS-Panel neben ALK weitere Marker wie z.B. ROS1 parallel mit zu testen, um eine qualifizierte Entscheidung für eine optimale Therapie zu ermöglichen

Síntese e caracterização de nanocompósitos magneto-poliméricos à base de polímeros extraídos de óleos naturais

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Nanociência e Nanobiotecnologia, 2015.O campo da Nanociência e Nanobiotecnologia desenvolve diversas pesquisas utilizando nanoestruturas com propriedades magnéticas, que podem apresentar diferentes propriedades físicas dependendo de seus compostos. Por isso podem apresentar diversas aplicações como: marcadores de fármacos, entrega de fármacos e microeletrônica. Este trabalho tem por objetivo sintetizar e caracterizar nanocompósito magneto-polimérico baseado em nanopartículas, com características superparamagnéticas, recobertas por polímero extraídos do óleo da semente de Carapa guianensis Aubl. Foram sintetizadas nanopartículas de óxidos de Ferro. O polímero é sintetizado utilizando o método de policondensação. O nanocompósito é preparado então pela dispersão das nanopartículas na matriz polimérica. Para caracterização das amostras são usadas diferentes técnicas como: Difração de Raios X, Microscopia Eletrônica de Transmissão, Microscopia Eletrônica de Varredura, Espectroscopia no UV-Vis, Espectroscopia no Infravermelho por Transformada de Fourier, Espectroscopia de Energia Dispersiva e Espectroscopia Fotoacústica. Os resultados indicam que a estrutura do nanocompósito magneto-poliméricos é do tipo core/shell onde o núcleo é formado por várias nanopartículas de γ-Fe2O3 recobertas pela matriz polimérica, que apresentou características do óleo natural utilizado em sua síntese.Nanoscience and Nanobiotechnology field develops several researches using nanostructures with magnetic properties, which may have different physical properties depending on their compounds. Therefore can be used in various applications as: drug markers, drug delivery and microelectronics. This study aims to synthesize and to characterize Magnetic- Polymeric Nanocomposites based on nanoparticles with superparamagnetic characteristics, coated with polymer oils extracted from seeds Carapa guianensis Aubl. Iron oxide nanoparticles were synthesized using the coprecipitation method. The polymer was synthesized using polycondensation method. Nanocomposite was prepared by dispersion of the nanoparticles in the polymer matrix. For characterization of the samples are used different techniques: X-ray Diffraction, Transmission Electron Microscopy, Scanning Electron Microscopy, UV-vis Spectroscopy, Fourier Transform Infrared Spectroscopy, Energy-dispersive X-Ray Spectroscopy and Photoacoustic Spectroscopy. The results indicate that magnetic-polymeric nanocomposites structure formed was type core/shell, wherein the core was formed by several nanoparticles of γ-Fe2O3, coated by the polymer matrix, which presents some characteristics of the natural oil used in their synthesi

The N-Terminus of Nef from HIV-1/SIV Associates with a Protein Complex Containing Lck and a Serine Kinase

AbstractThe Nef protein of human and primate lentiviruses is a key factor in HIV/SIV pathogenesis. Here we report that Nef associates with two different kinases, forming a multiprotein complex at the far N-terminus of the viral protein. One of the kinases was identified as Lck, whereas the second protein was found to be a serine kinase that phosphorylated Nef and Lck in vitro and could be discriminated from the serine kinase identified previously. The Nef-associated kinase complex (NAKC) was demonstrated in COS cells, in HIV- infected cells, and in vitro using recombinant Lck and Nef proteins. Deletion of a short amphipathic α-helix in the N-terminus, which was found to be conserved in all Nef proteins, inhibited association of the NAKC and significantly reduced virion infectivity

Synthesis, Characterization and Stabilization Analysis of Ferrofluid Based on Amazon Vegetable Copaifera O. L. Oil

The Nanoscience and Nanobiotechnology develops several researches using nanostructures with magnetic properties. These nanostructures can have different physical properties depending on their composition and therefore can be used in various applications such as: drug markers and carriers, microelectronics, magnetic separations and environmental applications. Furthermore, the use of natural components in the synthesis of these nanostructures may come to complement these nanomaterials due to their underexplored properties and their renewable origin. This research will aim to carry out a pre-study to analyze the stability of magnetic fluids based on Fe3O4 nanoparticles functionalized with vegetable oil from the Copaifera SPP plant, known as Copaiba. Therefore, the Fe3O4 nanoparticles will be synthesized by the coprecipitation method by hydrolysis in an alkaline solution. Ferrofluid will be prepared by dispersing magnetic nanoparticles, at different concentrations, in the “in natura” oil. For the samples characterization measurements were taken of: UV-vis, FTIR, NIR spectrophotometry, XRD and Magnetic Susceptibility, the temporal analysis was recorded periodically, observing the possible stability of the samples. This work reports that the ferrofluid showed good stability and the magnetic properties associated with the nanoparticles the properties of the Amazon oil used

Proposta de construção de um sistema de aquisição de dados para o estudo de movimentos em queda livre utilizando sensores indutivos em plataforma arduino / Proposal for the construction of a data acquisition system for the study of free fall movements using inductive sensors on an arduino platform

O uso de técnicas para aquisição de dados vem se intensificando e evoluindo junto com a tecnologia. Este trabalho tem como objetivo desenvolver um sistema para a aquisição de dados utilizando sensores indutivos em experimento de queda livre. Para o desenvolvimento do sistema, utilizara-se plataforma Arduíno, visto que é uma plataforma utilizada em aplicações industriais, automação e desenvolvimento de projetos. As vantagens de utilização do Arduíno encontra-se em uma plataforma barata e de fácil utilização. Para demonstrar o sistema, o mesmo foi aplicado ao estudo da aceleração da gravidade em queda livre, onde já existe uma solução comercial para aquisição  de dados, mas por conta de ser uma plataforma comercial, existe um alto custo para aquisição e manutenção desses equipamentos. Para confecção do projeto, foi utilizado uma placa Arduíno UNO R3 e sensores indutivos LJ12A3-4-Z/BX, sendo a parte estrutural, todas as peças impressas em 3D. Com o primeiro programa desenvolvido, foi realizado o primeiro protótipo, onde com os dados obtidos, o sistema se mostrou bastante confiável para realizar medições de tempo para cálculo de aceleração da gravidade e de baixo custo, em comparação com as soluções comercias, se mostrando uma solução barata e de fácil aquisição e manutenção, um dos pontos positivos no sistema desenvolvido é a possibilidade de atualizações e modificações, onde o sistema desenvolvido pode ser acoplado em nuvem para armazenamento de dados e controle remoto ou até um sistema de Database para comparação de medidas e armazenamento de medidas. Será apresentado o desenvolvimento deste sistema e os componentes necessários em caso de replicação para outras áreas

Diagnostic procedures for non-small-cell lung cancer (NSCLC): recommendations of the European Expert Group

Background There is currently no Europe-wide consensus on the appropriate preanalytical measures and workflow to optimise procedures for tissue-based molecular testing of non-small-cell lung cancer (NSCLC). To address this, a group of lung cancer experts (see list of authors) convened to discuss and propose standard operating procedures (SOPs) for NSCLC. Methods Based on earlier meetings and scientific expertise on lung cancer, a multidisciplinary group meeting was aligned. The aim was to include all relevant aspects concerning NSCLC diagnosis. After careful consideration, the following topics were selected and each was reviewed by the experts: surgical resection and sampling; biopsy procedures for analysis; preanalytical and other variables affecting quality of tissue; tissue conservation; testing procedures for epidermal growth factor receptor, anaplastic lymphoma kinase and ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) in lung tissue and cytological specimens; as well as standardised reporting and quality control (QC). Finally, an optimal workflow was described. Results Suggested optimal procedures and workflows are discussed in detail. The broad consensus was that the complex workflow presented can only be executed effectively by an interdisciplinary approach using a well-trained team. Conclusions To optimise diagnosis and treatment of patients with NSCLC, it is essential to establish SOPs that are adaptable to the local situation. In addition, a continuous QC system and a local multidisciplinary tumour-type-oriented board are essential

A Mechanistic, Model-Based Approach to Safety Assessment in Clinical Development

Peer reviewe

Supporting Youth Athletes during Covid-19: Guidance for parents and guardians.

This document aims to provide psychological guidance for parents and guardians of youth athletes during the current Covid-19 pandemic. Since lockdown (on 23 March 2020), and the subsequent emergence from lockdown in the United Kingdom (UK), youth athletes have been forced to significantly change their training, with the cancellation of formal training sessions and reintegration with social distancing. Throughout this time competitive sport was suspended, and in this transition phase, it is still unclear when all competitions will commence. These changes for some can lead to feelings of anxiety, frustration, and uncertainty. We offer psychological guidance to help parents and guardians maintain their own wellbeing, and support their children in their return to competitive sport. Three key priorities have been identified as critical for supporting youth athletes in managing the Covid-19 transition

KRASG12C/TP53 co-mutations identify long-term responders to first line palliative treatment with pembrolizumab monotherapy in PD-L1 high (≥50%) lung adenocarcinoma

Background: Pembrolizumab is a standard of care as first line palliative therapy in PD-L1 overexpressing (≥50%) non-small cell lung cancer (NSCLC). This study aimed at the identification of KRAS and TP53-defined mutational subgroups in the PD-L1 high population to distinguish long-term responders from those with limited benefit. Methods: In this retrospective, observational study, patients from 4 certified lung cancer centers in Berlin, Germany, having received pembrolizumab monotherapy as first line palliative treatment for lung adenocarcinoma (LuAD) from 2017 to 2018, with PD-L1 expression status and targeted NGS data available, were evaluated. Results: A total of 119 patients were included. Rates for KRAS, TP53 and combined mutations were 52.1%, 47.1% and 21.9%, respectively, with no association given between KRAS and TP53 mutations (P=0.24). By trend, PD-L1 expression was higher in KRAS-positive patients (75% vs. 65%, P=0.13). Objective response rate (ORR), median progression-free survival (PFS) and overall survival (OS) in the KRASG12C group (n=32, 51.6%) were 63.3%, 19.8 months (mo.) and not estimable (NE), respectively. Results in KRASother and wild type patients were similar and by far lower (42.7%, P=0.06; 6.2 mo., P<0.001; 23.4 mo., P=0.08). TP53 mutations alone had no impact on response and survival. However, KRASG12C/TP53 co-mutations (n=12) defined a subset of long-term responders (ORR 100.0%, PFS 33.3 mo., OS NE). In contrast, patients with KRASother/TP53 mutations showed a dismal prognosis (ORR 27.3%, P=0.002; PFS 3.9 mo., P=0.001, OS 9.7 mo., P=0.02). Conclusions: A comprehensive assessment of KRAS subtypes and TP53 mutations allows a highly relevant prognostic differentiation of patients with metastatic, PD-L1 high LuAD treated upfront with pembrolizumab

Mucosal melanomas of different anatomic sites share a common global DNA methylation profile with cutaneous melanoma but show location-dependent patterns of genetic and epigenetic alterations

Cutaneous, ocular, and mucosal melanomas are histologically indistinguishable tumors that are driven by a different spectrum of genetic alterations. With current methods, identification of the site of origin of a melanoma metastasis is challenging. DNA methylation profiling has shown promise for the identification of the site of tumor origin in various settings. Here we explore the DNA methylation landscape of melanomas from different sites and analyze if different melanoma origins can be distinguished by their epigenetic profile. We performed DNA methylation analysis, next generation DNA panel sequencing, and copy number analysis of 82 non-cutaneous and 25 cutaneous melanoma samples. We further analyzed eight normal melanocyte cell culture preparations. DNA methylation analysis separated uveal melanomas from melanomas of other primary sites. Mucosal, conjunctival, and cutaneous melanomas shared a common global DNA methylation profile. Still, we observed location-dependent DNA methylation differences in cancer-related genes, such as low frequencies of RARB (7/63) and CDKN2A promoter methylation (6/63) in mucosal melanomas, or a high frequency of APC promoter methylation in conjunctival melanomas (6/9). Furthermore, all investigated melanomas of the paranasal sinus showed loss of PTEN expression (9/9), mainly caused by promoter methylation. This was less frequently seen in melanomas of other sites (24/98). Copy number analysis revealed recurrent amplifications in mucosal melanomas, including chromosomes 4q, 5p, 11q and 12q. Most melanomas of the oral cavity showed gains of chromosome 5p with TERT amplification (8/10), while 11q amplifications were enriched in melanomas of the nasal cavity (7/16). In summary, mucosal, conjunctival, and cutaneous melanomas show a surprisingly similar global DNA methylation profile and identification of the site of origin by DNA methylation testing is likely not feasible. Still, our study demonstrates tumor location-dependent differences of promoter methylation frequencies in specific cancer-related genes together with tumor site-specific enrichment for specific chromosomal changes and genetic mutations. (c) 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland