Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

Background

Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.

Method

We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.

Results

Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.

Conclusion

Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.     

The Role of Personalised Choice in Decision Support: A Randomized Controlled Trial of an Online Decision Aid for Prostate Cancer Screening

ImportanceDecision support tools can assist people to apply population-based evidence on benefits and harms to individual health decisions. A key question is whether “personalising” choice within decisions aids leads to better decision quality.ObjectiveTo assess the effect of personalising the content of a decision aid for prostate cancer screening using the Prostate Specific Antigen (PSA) test.DesignRandomized controlled trial.SettingAustralia.Participants1,970 men aged 40–69 years were approached to participate in the trial.Intervention1,447 men were randomly allocated to either a standard decision aid with a fixed set of five attributes or a personalised decision aid with choice over the inclusion of up to 10 attributes.Results5% of men in the fixed attribute group scored ‘Have a PSA test’ as the opinion generated by the aid, as compared to 62% of men in the personalised choice group (χ² = 569.38, 2df, p< 0001). Those men who used the personalised decision aid had slightly higher decision quality (t = 2.157, df = 1444, p = 0.031). The men in the personalised choice group made extensive use of the additional decision attributes. There was no difference between the two groups in terms of their stated intention to undergo screening in the next 12 months.ConclusionsTogether, these findings suggest that personalised decision support systems could be an important development in shared decision-making and patient-centered care.

Trial Registration

Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12612000723886     

The Impacts of Using Smartphone Dating Applications on Sexual Risk Behaviours in College Students in Hong Kong

Dating applications (apps) on smartphones have become increasingly popular. The aim of this study was to explore the association between the use of dating apps and risky sexual behaviours. Data were collected in four university campuses in Hong Kong. Subjects completed a structured questionnaire asking about the use of dating apps, sexual behaviours, and sociodemographics. Multiple linear and logistics regressions were used to explore factors associated with sexual risk behaviours. Six hundred sixty-six subjects were included in the data analysis. Factors associated with having unprotected sexual intercourse with more lifetime sexual partners included use of dating apps (β = 0.93, p<0.01), having one’s first sexual intercourse before 16 years of age (β = 1.74, p<0.01), being older (β = 0.4, p<0.01), currently being in a relationship (= 0.69, p<0.05), having a monthly income at least HKD$5,000 (β = 1.34, p<0.01), being a current smoker (β = 1.52, p<0.01), and being a current drinker (β = 0.7, p<0.01). The results of a multiple logistic regression analysis found that users of dating apps (adjust odds ratio: 0.52, p<0.05) and current drinkers (adjust odds ratio: 0.40, p<0.01) were less likely to have consistent condom use. Users of dating apps (adjust odds ratio: 1.93, p<0.05), bisexual/homosexual subjects (adjust odds ratio: 2.57, p<0.01) and female subjects (adjust odds ratio: 2.00, p<0.05) were more likely not to have used condoms the last time they had sexual intercourse. The present study found a robust association between using dating apps and sexual risk behaviours, suggesting that app users had greater sexual risks. Interventions that can target app users so that they can stay safe when seeking sexual partners through dating apps should be developed.     

Cuticle Thickening in a Pyrethroid-Resistant Strain of the Common Bed Bug,Cimex lectularius L. (Hemiptera: Cimicidae)

Thickening of the integument as a mechanism of resistance to insecticides is a well recognised phenomenon in the insect world and, in recent times, has been found in insects exhibiting pyrethroid-resistance. Resistance to pyrethroid insecticides in the common bed bug, Cimex lectularius L., is widespread and has been frequently inferred as a reason for the pest’s resurgence. Overexpression of cuticle depositing proteins has been demonstrated in pyrethroid-resistant bed bugs although, to date, no morphological analysis of the cuticle has been undertaken in order to confirm a phenotypic link. This paper describes examination of the cuticle thickness of a highly pyrethroid-resistant field strain collected in Sydney, Australia, in response to time-to-knockdown upon forced exposure to a pyrethroid insecticide. Mean cuticle thickness was positively correlated to time-to-knockdown, with significant differences observed between bugs knocked-down at 2 hours, 4 hours, and those still unaffected at 24 hours. Further analysis also demonstrated that the 24 hours survivors possessed a statistically significantly thicker cuticle when compared to a pyrethroid-susceptible strain of C. lectularius. This study demonstrates that cuticle thickening is present within a pyrethroid-resistant strain of C. lectularius and that, even within a stable resistant strain, cuticle thickness will vary according to time-to-knockdown upon exposure to an insecticide. This response should thus be considered in future studies on the cuticle of insecticide-resistant bed bugs and, potentially, other insects.     

The nature of plant adaptations to salinity stress has trophic consequences

In different ecosystems herbivores highly prefer particular plant species. This is often explained in a stoichiometric framework of nutrient‐based plant adaptations to herbivory. We hypothesize that such super‐palatability can also arise as an evolutionary by‐product of osmoregulatory adaptations of plants to stressful environmental conditions, as salinity, drought and cold. Here, we investigate in a coastal salt marsh why some plant species are highly preferred by migratory brent geese Branta bernicla bernicla in spring while others are avoided. This salt marsh is an important spring staging site for the geese. Sufficient energy storage in a short period is critical to enable their northward migration to Siberia and subsequent reproduction. We test if geese prefer plants that balance their internal osmotic potential with the saline environment through energy‐rich soluble sugars over plant species that use (compartmentalized) salts for this. We find that plant nitrogen and acid detergent fiber content, classic predictors of herbivore preferences, poorly explain which plants the geese prefer. Instead, plant species that are highly preferred by the geese adapt to salinity by high soluble sugar concentrations while avoided species do this by high plant salt concentrations. Thus, the type of osmoregulatory adaptation to stress displayed by different plant species is a good predictor for the food preference of geese on this salt marsh. We suggest that variation in other types of osmoregulation‐based stress adaptations, as plant cold adaptations in tundras and plant drought adaptations in savannas, have similar important consequences for trophic interactions.     

Lifelong choline supplementation ameliorates Alzheimer’s disease pathology and associated cognitive deficits by attenuating microglia activation

Currently, there are no effective therapies to ameliorate the pathological progression of Alzheimer's disease (AD). Evidence suggests that environmental factors may contribute to AD. Notably, dietary nutrients are suggested to play a key role in mediating mechanisms associated with brain function. Choline is a B‐like vitamin nutrient found in common foods that is important in various cell functions. It serves as a methyl donor and as a precursor for production of cell membranes. Choline is also the precursor for acetylcholine, a neurotransmitter which activates the alpha7 nicotinic acetylcholine receptor (α7nAchR), and also acts as an agonist for the Sigma‐1 R (σ1R). These receptors regulate CNS immune response, and their dysregulation contributes to AD pathogenesis. Here, we tested whether dietary choline supplementation throughout life reduces AD‐like pathology and rescues memory deficits in the APP/PS1 mouse model of AD. We exposed female APP/PS1 and NonTg mice to either a control choline (1.1 g/kg choline chloride) or a choline‐supplemented diet (5.0 g/kg choline chloride) from 2.5 to 10 months of age. Mice were tested in the Morris water maze to assess spatial memory followed by neuropathological evaluation. Lifelong choline supplementation significantly reduced amyloid‐β plaque load and improved spatial memory in APP/PS1 mice. Mechanistically, these changes were linked to a decrease of the amyloidogenic processing of APP, reductions in disease‐associated microglial activation, and a downregulation of the α7nAch and σ1 receptors. Our results demonstrate that lifelong choline supplementation produces profound benefits and suggest that simply modifying diet throughout life may reduce AD pathology.     

A general framework for propagule dispersal in mangroves

Dispersal allows species to shift their distributions in response to changing climate conditions. As a result, dispersal is considered a key process contributing to a species' long‐term persistence. For many passive dispersers, fluid dynamics of wind and water fuel these movements and different species have developed remarkable adaptations for utilizing this energy to reach and colonize suitable habitats. The seafaring propagules (fruits and seeds) of mangroves represent an excellent example of such passive dispersal. Mangroves are halophytic woody plants that grow in the intertidal zones along tropical and subtropical shorelines and produce hydrochorous propagules with high dispersal potential. This results in exceptionally large coastal ranges across vast expanses of ocean and allows species to shift geographically and track the conditions to which they are adapted. This is particularly relevant given the challenges presented by rapid sea‐level rise, higher frequency and intensity of storms, and changes in regional precipitation and temperature regimes. However, despite its importance, the underlying drivers of mangrove dispersal have typically been studied in isolation, and a conceptual synthesis of mangrove oceanic dispersal across spatial scales is lacking. Here, we review current knowledge on mangrove propagule dispersal across the various stages of the dispersal process. Using a general framework, we outline the mechanisms and ecological processes that are known to modulate the spatial patterns of mangrove dispersal. We show that important dispersal factors remain understudied and that adequate empirical data on the determinants of dispersal are missing for most mangrove species. This review particularly aims to provide a baseline for developing future research agendas and field campaigns, filling current knowledge gaps and increasing our understanding of the processes that shape global mangrove distributions.