Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.

AIMS: Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. METHODS: 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. RESULTS: Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p

A new generation of companion diagnostics: cobas BRAF, KRAS and EGFR mutation detection tests

The cobas® (Roche) portfolio of companion diagnostics in oncology currently has three assays CE-marked for in vitro diagnostics. Two of these (EGFR and BRAF) are also US FDA-approved. These assays detect clinically relevant mutations that are correlated with response (BRAF, EGFR) or lack of response (KRAS) to targeted therapies such as selective mutant BRAF inhibitors in malignant melanoma, tyrosine kinases inhibitor in non-small cell lung cancer and anti-EGFR monoclonal antibodies in colorectal cancer, respectively. All these assays are run on a single platform using DNA extracted from a single 5 µm section of a formalin-fixed paraffin-embedded tissue block. The assays provide an ‘end-to-end’ solution from extraction of DNA to automated analysis and report on the cobas z 480. The cobas tests have shown robust and reproducible performance, with high sensitivity and specificity and low limit of detection, making them suitable as companion diagnostics for clinical use.

Ajustamento Mental ao Cancro do Pulmão: o papel da autocompaixão e do suporte social

A presente investigação pretendeu ver cumpridos três dos principais objetivos: 1) Estudar as variáveis sociodemográficas e clínicas que caracterizam os doentes com cancro do pulmão; 2) Explorar a relação entre o ajustamento mental ao cancro do pulmão, a autocompaixão, o suporte social e os estados emocionais negativos dos doentes; 3) Examinar o impacto da autocompaixão e do suporte social em relação ao ajustamento mental e aos estados emocionais negativos em doentes com cancro do pulmão. A amostra é constituída por 55 indivíduos (38 homens e 17 mulheres) diagnosticados com cancro do pulmão e com idades compreendidas entre os 44 e os 87 anos, acompanhados medicamente no Hospital de Dia de Oncologia do Centro Hospitalar e Universitário de Coimbra. Como instrumentos de medida para avaliar o ajustamento mental ao cancro, a autocompaixão, o suporte social e os estados emocionais negativos dos participantes foram utilizadas a Escala de Ajustamento Mental ao Cancro (MiniMac), a Escala de Autocompaixão (Selfcs), a Escala de Satisfação com o Suporte Social (Esss) e a Escala de Sintomatologia Psicopatológica (Eads-21). Os resultados obtidos revelaram uma associação significativa entre algumas variáveis clínicas, nomeadamente ser fumador, perceção da gravidade da doença, existência de antecedentes familiares com doença oncológica, e as variáveis em estudo (ajustamento mental, autocompaixão, suporte social e psicopatologia). Foram ainda encontradas correlações significativas entre o ajustamento mental e as estratégias de regulação emocional (autocompaixão), suporte social e psicopatologia. Por último, as análises de regressão linear múltipla mostraram que o modelo preditor da sintomatologia depressiva e do ajustamento mental (avaliado pela dimensão de desânimo) inclui o mindfulness como um preditor significativo. Já em relação ao modelo preditor do stress, o grau de satisfação com o suporte dos amigos revelou ser um contributo importante. Estes resultados têm implicações práticas, sugerindo que estes doentes podem no seu programa terapêutico beneficiar do desenvolvimento deste tipo de estratégias (novas formas de se relacionarem com as suas experiências emocionais e qualidade das suas redes sociais) no sentido de promover um melhor ajustamento mental à sua condição. / The current investigation intended to study three main objetives: 1) to study the sociodemographical and clinical variables which characterize those who suffer from lung cancer; 2) to explore the relation between the mental adjustment to lung cancer, selfcompassion, social support and the negative mental conditions of the sick person; 3) to analyse the impact of self-compassion and the social support in relation to the mental adjustment and to the negative mental conditions of a sick person with lung cancer. The sample is made of 55 individuals (38 males and 17 females) diagnosed with lung cancer, aged between 44 and 87 years old, using medicines at the Hospital de Dia de Oncologia do Centro Hospitalar e Universitário de Coimbra. The Mini Mental Adjustment to Cancer Scale (MiniMac), the Self-Compassion Scale (Selfcs), the Escala de Satisfação com o Suporte Social (Esss) and the Depression Anxiety Stress Scales (Eads-21) scales were used as measuring instruments of evaluation of the mental adjustment to lung cancer, selfcompassion, social support and the negative mental conditions of the sick person. The results revealed a significant association between some clinical variables (being a smoker, awareness of the gravity of sickness, precedent relatives who suffered from cancer) and the variables in study (mental adjustment, self-compassion, social support and psychopathology). There were also found significant correlations between mental adjustment and the strategies used for emotional adjustment (self-compassion), the social support and the psychopathology. At last the multiple linear regretting analysis has shown that the predictor model of depressive symptomatology and the mental adjustment (analysed by the discouragement dimension) includes mindfulness as a significant predictor. However in what concerns to the stress model predictor, the satisfaction level with friends support revealed itself has being of high importance. These results have practical consequences, suggesting that sick people can benefit in their therapeutic program of these kind of strategies (new ways of leading with their emotional experiences and the quality of their social relationships) so they can promote a better mental adjustment to their health condition.

AN ECONOMIC ANALYSIS OF THE ADDITION OF BEVACIZUMAB TO STANDARD CHEMOTHERAPY IN PATIENTS WITH MALIGNANT PLEURAL MESOTHELIOMA

Thesis (Master's)--University of Washington, 2016-08

Adoecimento osteomuscular de trabalhadores portuários avulsos e o processamento do raciocínio clínico da enfermagem

Introdução: O trabalho portuário é composto por condicionantes socioambientais necessários à manutenção das funções operativas, mas que influenciam na produção de doenças osteomusculares. O conhecimento desses condicionantes instrumentaliza o raciocínio clínico da Enfermagem para o planejamento de ações em saúde. Desta forma, defende-se a tese de que “O conhecimento dos condicionantes socioambientais e pessoais do adoecimento osteomuscular do trabalhador portuário avulso fornece elementos ao processamento do raciocínio clínico da Enfermagem, para assistência em saúde do trabalhador”. Objetivos: identificar evidências científicas de adoecimento ocupacional do trabalhador portuário publicadas na literatura cientifica; caracterizar o tipo, a localização e a intensidade de sintomas osteomusculares relacionados com os condicionantes socioambientais do trabalho portuário; Relacionar as doenças osteomusculares autorreferidas por trabalhadores portuários e os condicionantes socioambientais deste trabalho. Percurso Metodológico: o estudo apresentou revisão sistemática, fundamentada no método Cochrane; e estudos descritivos e exploratórios de abordagem quantitativa, realizado por meio de entrevista semi-estruturada com 232 trabalhadores portuários avulsos. Os dados foram analisados no software Statistical Package for the Social Sciences (SPSS) 21.0, por frequência simples, proporções e testes inferenciais não-paramétricos. A tese integra o macro projeto de pesquisa “Saúde do Trabalhador, Riscos, Acidentes e Doenças Relacionadas ao Trabalho: Estudo com Trabalhadores em um Porto no Extremo Sul do Brasil”, aprovado pelo Comitê de Ética em Pesquisa da Universidade Federal do Rio Grande (CEPAS-FURG) sob parecer número 118/2013. Resultados: Na revisão sistemática, selecionaram-se 16 publicações; todas as publicações pertenceram ao nível de evidência quatro, destacando o câncer pulmonar, doenças osteomusculares e isquêmicas, com nexo causal em riscos químicos oriundos da exaustão veicular e das cargas transportadas. Nos estudos descritivos, os sintomas prevalentes foram a dor leve em membros superiores (51,7%) e intensa a insuportável na coluna vertebral (19%). Os dois adoecimentos mais autorreferidos foram lombocitalgia (36,8%; n=50 – em terra e 28,1%; n=27 – a bordo) e tendinite (27,9% - em terra e 31,3% - a bordo). Discussão: O câncer pulmonar ocupacional foi causado por componentes químicos da exaustão veicular e do amianto transportado nas operações portuárias. Com relação à saúde muscular, a idade, o tempo e a jornada de trabalho mostraram-se condicionantes importantes na identificação de sintomas e adoecimentos, e o quanto estes fatores interveem na percepção da intensidade, contribuindo no autocuidado para prevenção e tratamento. Conclusão: O conhecimento dos condicionantes socioambientais relacionados ao trabalhador e caracterizados nos ambientes de trabalho deve ser atual e pregresso, o que somado à apreensão dos sintomas e adoecimentos autorreferidos pelos trabalhadores instrumentalizou o RC, identificando uma atuação profissional em longo prazo para dirimir os adoecimentos identificados. As características clínicas obtidas, em conjunto com a literatura, conduziram ao processamento do RC da enfermagem nesta realidade, sendo a informação em saúde um ponto chave para a promoção da saúde muscular dos trabalhadores.

Medidas de concentração de radônio proveniente de argamassas de cimento portland, gesso e fosfogesso

Portland cement being very common construction material has in its composition the natural gypsum. To decrease the costs of manufacturing, the cement industry is substituting the gypsum in its composition by small quantities of phosphogypsum, which is the residue generated by the production of fertilizers and consists essentially of calcium dihydrate and some impurities, such as fluoride, metals in general, and radionuclides. Currently, tons of phosphogypsum are stored in the open air near the fertilizer industries, causing contamination of the environment. The 226 Ra present in these materials, when undergoes radioactive decay, produces the 222Rn gas. This radioactive gas, when inhaled together with its decay products deposited in the lungs, produces the exposure to radiation and can be a potential cause of lung cancer. Thus, the objective of this study was to measure the concentration levels of 222Rn from cylindrical samples of Portland cement, gypsum and phosphogypsum mortar from the state of Paraná, as well as characterizer the material and estimate the radon concentration in an environment of hypothetical dwelling with walls covered by such materials. Experimental setup of 222Rn activity measurements was based on AlphaGUARD detector (Saphymo GmbH). The qualitative and quantitative analysis was performed by gamma spectrometry and EDXRF with Au and Ag targets tubes (AMPTEK), and Mo target (ARTAX) and mechanical testing with x- ray equipment (Gilardoni) and the mechanical press (EMIC). Obtained average values of radon activity from studied materials in the air of containers were of 854 ± 23 Bq/m3, 60,0 ± 7,2 Bq/m3 e 52,9 ± 5,4 Bq/m3 for Portland cement, gypsum and phosphogypsum mortar, respectively. These results extrapolated into the volume of hypothetical dwelling of 36 m3 with the walls covered by such materials were of 3366 ± 91 Bq/m3, 237 ± 28 Bq/m3 e 208 ± 21 Bq/m3for Portland cement, gypsum and phosphogypsum mortar, respectively. Considering the limit of 300 Bq/m3 established by the ICRP, it could be concluded that the use of Portland cement plaster in dwellings is not secure and requires some specific mitigation procedure. Using the results of gamma spectrometry there were calculated the values of radium equivalent activity concentrations (Raeq) for Portland cement, gypsum and phosphogypsum mortar, which were obtained equal to 78,2 ± 0,9 Bq/kg; 58,2 ± 0,9 Bq/kg e 68,2 ± 0,9 Bq/kg, respectively. All values of radium equivalent activity concentrations for studied samples are below the maximum level of 370 Bq/kg. The qualitative and quantitative analysis of EDXRF spectra obtained with studied mortar samples allowed to evaluate quantitate and the elements that constitute the material such as Ca, S, Fe, and others.

The broad spectrum of bioactive properties of phenolic extracts: a prospective study in three different plants

Natural resources like plants are currently used all over developed and under developed countries of the world as traditional home remedies and are promising agents for drug discovery as they play crucial role in traditional medicine. The use of plants for medicinal purpose usually varies from country to country and region to region because their use depends on the history, culture, philosophy and personal attitudes of the users (Ahmad et al., 2015). The use of plants and plant products as drugs predates the written human history (Hayta et al., 2014). Plants are a very important resource for traditional drugs and around 80% of the population of the planet use plants for the treatment of many diseases and traditional herbal medicine accounts for 30-50% of the total medicinal consumption in China. In North America, Europe and other well-developed regions over 50% of the population have used traditional preparations at least once (Dos Santos Reinaldo et al., 2015). Medicinal plants have been used over years for multiple purposes, and have increasingly attract the interest of researchers in order to evaluate their contribution to health maintenance and disease’s prevention (Murray, 2004). Recently between 50,000 and 70,000 species of plants are known and are being used in the development of modern drugs. Plants were the main therapeutic agents used by humans from the 19th century, and their role in medicine is always topical (Hayta et al., 2014). The studies of medicinal plants are rapidly increasing due to the search for new active molecules, and to improve the production of plants or bioactive molecules for the pharmaceutical industries (Rates, 2001). Several studies have been reported, but numerous active compounds directly responsible for the observed bioactive properties remain unknown, while in other cases the mechanism of action is not fully understood. According to the WHO 25% of all modern medicines including both western and traditional medicine have been extracted from plants, while 75% of new drugs against infective diseases that have arrived between 1981 and 2002 originated from natural sources, it was reported that the world market for herbal medicines stood at over US $60 billion per year and is growing steadily (Bedoya et al., 2009). Traditional medicine has an important economic impact in the 21st century as it is used worldwide, taking advantage on the low cost, accessibility, flexibility and diversity of medicinal plants (Balunas & Kinghorn, 2005).

Study of antioxidant, antiproliferative and apoptosis-inducing properties of wild mushrooms from the Northeast of Portugal

Mushrooms are known as a powerful source of bioactive compounds including antioxidants, inhibitors of human tumour cell lines growth, inducers of apoptosis and enhancers of immunity. Indeed, many pre-clinical studies have been conducted in human tumour cell lines and in some cases a number of compounds isolated from mushrooms have followed to clinical trials. The Northeast of Portugal is one of the European regions with higher wild mushrooms diversity. However, to our knowledge, no studies had been conducted so far to verify their bioactivities. The main aim of this work was the evaluation of the bioactive properties (antioxidant properties and growth inhibitory potential on human tumour cell lines) of wild edible mushrooms collected in the Northeast of Portugal. Once properly identified, methanolic, ethanolic and boiling water extracts were prepared from thirty eight wild mushroom species collected in that region. Chemical characterization was obtained by high performance liquid chromatography (HPLC) coupled to a photodiode array detector (DAD) or to a refraction index detector (RI). Antioxidant activity assays were carried out in those extracts, including evaluation of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals scavenging capacity, reducing power and inhibition of β-carotene bleaching. Extract-induced cell growth inhibition was assessed with the sulforhodamine B assay in four human tumour cell lines (NCI-H460 - lung cancer, MCF-7 -breast cancer, HCT-15 -colon cancer and AGS - gastric cancer). The effects on cell cycle profile and apoptosis were evaluated by flow cytometry and the effect on the expression levels of proteins related to cell cycle and apoptosis was further investigated by Western blotting. Three wild edible mushroom species revealed growth inhibitory activity in the studied human tumour cell lines: Clitocybe alexandri ethanolic extract, Lepista inversa methanolic extract and Suillus collinitus methanolic extract. C. alexandri ethanolic extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells, in the NCI-H460 cell line. The analysed mushroom species also provided interesting antioxidant potential, mainly the boiling water extract of L. inversa which showed the highest DPPH radical scavenging activity, reducing power and β-carotene bleaching inhibition. S. collinitus methanolic extract induced a slight increase in the number of cells in G1, with a concomitant decrease in the percentage of cells in the S phase of the cell cycle and an increase in the percentage of apoptotic cells, in the MCF-7 cell line. The combined use of the S. collinitus methanolic extract and etoposide caused a greater decrease in the percentage of cell growth, when compared to either of them used individually, indicating the potential benefit of this combination. The tested extracts were chemically characterized and protocatechuic, p-hydroxybenzoic, p-coumaric and cinnamic acids were the main compounds identified on the phenolic (methanolic and ethanolic) extracts, while mannitol, trehalose and arabinose were the main sugars found in the polysaccharidic (boiling water) extracts after hydrolysis. The individual compounds identified in the extracts were submitted to a screening of tumour cells growth inhibitory activity, but only the phenolic acids and a related compound, cinnamic acid, presented activity. This compound was found to be the most potent one regarding cell growth inhibition in the NCI-H460 cell line. The effect of the individual and combined treatment with the identified compounds was also evaluated. Cinnamic and protochatequic acids caused a statistically significantly reduction in the number of viable cells. In addition, p-hydroxybenzoic acid did not show any significantly reduction in the viable cell number. Nevertheless, it was verified that the concomitant use of the three compounds provided the strongest decrease in the viable cell number, suggesting a possible concomitant effect of those compounds. Overall, the present work has contributed to further understand the bioactive potential of wild edible mushrooms from the Northeast of Portugal. This study allowed to identify some species with antioxidant or tumour cell growth inhibitory potential.

A prospective study on bioactive properties of wild mushrooms mycelium grown in vitro under different conditions

Wild mushrooms have been extensively studied for their value as sources of high quality nutrients and of powerful physiologically bioactive compounds [1,2]. The present study was designed to evaluate the in vitro development of two wild edible mushroom species: Pleurotus eryngii (DC.) Quél. and Suillus belinii (Inzenga) Watling, by testing different solid (Potato Dextrose Agar medium –PDA and Melin-Norkans medium- MMN) and liquid culture media (Potato dextrose broth- PDB and Melin-Norkans medium- MMN). Each strain of mushroom produces a special type of mycelium and this range of characteristics varies in form, color and growth rate. S. bellinii presents a pigmented and rhizomorphic mycelia, whereas, P. eryngii has depigmented and cottony mycelia. The mycelium isolated and grown in PDA showed a faster radial growth compared to the mycelium isolated and grown in both solid and liquid incomplete MMN medium. P. eryngii exhibited a rapid growth and a higher mycelia biomass in both medium compared to S. belinii. Moreover, the obtained mycelia will be characterized in terms of well-recognized bioactive compounds namely, phenolic acids and mycosterols (mainly ergosterol), by using high performance liquid chromatography coupled to diode array and ultraviolet detectors, respectively. These compounds will be correlated to mycelia bioactivity: i) antioxidant activity, evaluated through free radicals scavenging activity, reducing power and lipid peroxidation inhibition in vitro assays; ii) anti-inflammatory activity, assessed through nitric oxide production inhibition in murine macrophages (RAW 264.7 cell line); iii) cytotoxic activity, evaluated either in human tumor cell lines (MCF-7- breast adenocarcinoma, NCIH460- non-small cell lung cancer, HeLa- cervical carcinoma and HepG2- hepatocellular carcinoma) as also in a non-tumor porcine primary liver cells culture established in-house (PLP2). Overall, our expectation is that the bioactive formulations obtained by in vitro culture can be applied as nutraceuticals or incorporated in functional foods.

HPLC-DAD-ESI/MS phenolic characterization and biological activity of Equisetum giganteum L.

Naturally-occurring phytochemicals have received a pivotal attention in the last years, due to the increasing evidences of biological activities. Equisetum giganteum L., commonly known as “giant horsetail”, is a native plant from Central and South America, being largely used in dietary supplements as diuretic, hemostatic, antiinflammatory and anti-rheumatic agents [1,2]. The aim of the present study was to evaluate the antioxidant (scavenging effects on 2,2-diphenyl-1-picrylhydrazyl radicals- RSA, reducing power- RP, β-carotene bleaching inhibition- CBI and lipid peroxidation inhibition- LPI), anti-inflammatory (inhibition of NO production in lipopolysaccharidestimulated RAW 264.7 macrophages) and cytotoxic (in a panel of four human tumor cell lines: MCF-7- breast adenocarcinoma, NCI-H460- non-small cell lung cancer, HeLa- cervical carcinoma and HepG2- hepatocellular carcinoma; and in non-tumor porcine liver primary cells- PLP2) properties of E. giganteum, providing a phytochemical characterization of its extract (ethanol/water, 80:20, v/v), by using highperformance liquid chromatography coupled to diode array detection and electrospray ionisation mass spectrometry (HPLC-DAD–ESI/MS). E. giganteum presented fourteen phenolic compounds, two phenolic acids and twelve flavonol glycoside derivatives, mainly kaempferol derivatives, accounting to 81% of the total phenolic content, being kaempferol-O-glucoside-O-rutinoside, the most abundant molecule (7.6 mg/g extract). The extract exhibited antioxidant (EC50 values = 123, 136, 202 and 57.4 μg/mL for RSA, RP, CBI and LPI, respectively), anti-inflammatory (EC50 value = 239 μg/mL) and cytotoxic (GI50 values = 250, 258, 268 and 239 μg/mL for MCF-7, NCI-H460, HeLa and HepG2, respectively) properties, which were positively correlated with its concentration in phenolic compounds. Furthermore, up to 400 μg/mL, it did not revealed toxicity in non-tumor liver cells. Thus, this study highlights the potential of E. giganteum extracts as rich sources of phenolic compounds that can be used in the food, pharmaceutical and cosmetic fields.

The impact of gamma irradiation on the cytotoxic properties and phenolic composition of Thymus vulgaris L. and Menta x piperita L.

Irradiation has been increasingly recognized as an effective decontamination technique, also ensuring the chemical and organoleptic quality of medicinal and aromatic plants 1 . The use of medicinal plants in the prevention and or treatment of several diseases has revealed satisfactory results as anti-inflammatory, antimutagenic, anti-cancer and antioxidant agents 2 . The aim of the present study was to evaluate the effects of gamma irradiation on the cytotoxic properties and phenolic composition of Thymus vulgaris L. and Menta x piperita L. (methanolic extracts). Phenolic compounds were analyzed by HPLC-DAD-ESI MS, while the cytotoxicity of the samples was assessed in MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma) cell lines, as also in non-tumor cells (PLP2). Thirteen and fourteen phenolic compounds were detected in T. vulgaris and M. piperita, respectively, but none of them was affected by the irradiation up to a dose of 10 kGy. However, despite there were no changes in the cytotoxic properties of irradiated peppermint samples in tumor cell lines, the thyme samples irradiated with 10 kGy showed higher cytotoxicity in comparison with the samples submitted to other doses (2 and 5 kGy). This highlights that 10 kGy can be a suitable dose to ensure the sanitary treatment, without modifying the bioactive composition and properties of these aromatic plants.