Recent advances in methodology for clinical trials in small populations : the InSPiRe project

Where there are a limited number of patients, such as in a rare disease, clinical trials in these small populations present several challenges, including statistical issues. This led to an EU FP7 call for proposals in 2013. One of the three projects funded was the Innovative Methodology for Small Populations Research (InSPiRe) project. This paper summarizes the main results of the project, which was completed in 2017. The InSPiRe project has led to development of novel statistical methodology for clinical trials in small populations in four areas. We have explored new decision-making methods for small population clinical trials using a Bayesian decision-theoretic framework to compare costs with potential benefits, developed approaches for targeted treatment trials, enabling simultaneous identification of subgroups and confirmation of treatment effect for these patients, worked on early phase clinical trial design and on extrapolation from adult to pediatric studies, developing methods to enable use of pharmacokinetics and pharmacodynamics data, and also developed improved robust meta-analysis methods for a small number of trials to support the planning, analysis and interpretation of a trial as well as enabling extrapolation between patient groups. In addition to scientific publications, we have contributed to regulatory guidance and produced free software in order to facilitate implementation of the novel methods

The Second Intron Is Essential for the Transcriptional Control of the Arabidopsis thaliana GLABRA3 Gene in Leaves

The GLABRA3 gene is a major regulator of trichome patterning in Arabidopsis thaliana. The regulatory regions important for the trichome-specific expression of GL3 have not been characterized yet. In this study, we used a combination of marker and rescue constructs to determine the relevant promoter regions. We demonstrate that a 1 kb 5' region combined with the second intron is sufficient to rescue the trichome mutant phenotype of gl3 egl3 mutants. Swap experiments of the second intron suggest that it is not sufficient to generally enhance the expression level of GL3. This implies that the second intron contains regulatory regions for the temporal and spatial regulation of GL3. The corresponding GUS-marker constructs revealed trichome-specific expression in young trichomes

Robustness of testing procedures for confirmatory subpopulation analyses based on a continuous biomarker

With the advent of personalized medicine, clinical trials studying treatment effects in subpopulations are receiving increasing attention. The objectives of such studies are, besides demonstrating a treatment effect in the overall population, to identify subpopulations, based on biomarkers, where the treatment has a beneficial effect. Continuous biomarkers are often dichotomized using a threshold to define two subpopulations with low and high biomarker levels. If there is insufficient information on the dependence structure of the outcome on the biomarker, several thresholds may be investigated. The nested structure of such subpopulations is similar to the structure in group sequential trials. Therefore, it has been proposed to use the corresponding critical boundaries to test such nested subpopulations. We show that for biomarkers with a prognostic effect that is not adjusted for in the statistical model, the variability of the outcome may vary across subpopulations which may lead to an inflation of the family-wise type 1 error rate. Using simulations we quantify the potential inflation of testing procedures based on group sequential designs. Furthermore, alternative hypotheses tests that control the family-wise type 1 error rate under minimal assumptions are proposed. The methodological approaches are illustrated by a trial in depression.peerReviewe

Marker Removal in Staphylococci via Cre Recombinase and Different lox Sites▿

Allelic replacement in staphylococci is frequently aided by antibiotic resistance markers that replace the gene(s) of interest. In multiply modified strains, the number of mutated genes usually correlates with the number of selection markers in the strain's chromosome. Site-specific recombination systems are capable of eliminating such markers, if they are flanked by recombinase recognition sites. In this study, a Cre-lox setting was established that allowed the efficient removal of resistance genes from the genomes of Staphylococcus carnosus and S. aureus. Two cassettes conferring resistance to erythromycin or kanamycin were flanked with wild-type or mutant lox sites, respectively, and used to delete single genes and an entire operon. After transformation of the cells with a newly constructed cre expression plasmid (pRAB1), genomic eviction of the resistance genes was observed in approximately one out of ten candidates analyzed and subsequently verified by PCR. Due to its thermosensitive origin of replication, the plasmid was then easily eliminated at nonpermissive temperatures. We anticipate that the system presented here will prove useful for generating markerless deletion mutants in staphylococci

Factors influencing the frequency of airway infections in underage refugees: a retrospective, cross sectional study

Background: Infections are a leading cause of refugee morbidity. Recent data on the rate of airway infections and factors influencing their spread in refugee reception centers is scarce. Methods: A retrospective, cross-sectional study of de-identified medical records with a focus on respiratory infections in underage refugees was conducted at two large German refugee reception centers. Results: In total, medical data from n = 10,431 refugees over an observational period of n = 819 days was analyzed. Among pediatric patients (n = 4289), 55.3% presented at least once to the on-site medical ward with an acute respiratory infection or signs thereof. In 38.4% of pediatric consultations, acute airway infections or signs thereof were present. Airway infections spiked during colder months and were significantly more prevalent amongst preschool and resettled children. Their frequency displayed a positive correlation with the number of refugees housed at the reception centers. Conclusions: We show that respiratory infections are a leading cause for morbidity in young refugees and that their rate is influenced age, season, status, and residential density. This illustrates the need to protect refugee children from contracting airway infections which may also reduce the spread of coronavirus disease 2019 (COVID-19) during the current pandemic

Supplemental material for Robustness of testing procedures for confirmatory subpopulation analyses based on a continuous biomarker

<p>Supplemental Material for Robustness of testing procedures for confirmatory subpopulation analyses based on a continuous biomarker by Alexandra Christine Graf, Gernot Wassmer, Tim Friede, Roland Gerard Gera and Martin Posch in Statistical Methods in Medical Research</p

Characterisation of Lipoma-Preferred Partner as a Novel Mechanotransducer in Vascular Smooth Muscle Cells

In arteries and arterioles, a chronic increase in blood pressure raises wall tension. This continuous biomechanical strain causes a change in gene expression in vascular smooth muscle cells (VSMCs) that may lead to pathological changes. Here we have characterised the functional properties of lipoma-preferred partner (LPP), a Lin11–Isl1–Mec3 (LIM)-domain protein, which is most closely related to the mechanotransducer zyxin but selectively expressed by smooth muscle cells, including VSMCs in adult mice. VSMCs isolated from the aorta of LPP knockout (LPP-KO) mice displayed a higher rate of proliferation than their wildtype (WT) counterparts, and when cultured as three-dimensional spheroids, they revealed a higher expression of the proliferation marker Ki 67 and showed greater invasion into a collagen gel. Accordingly, the gelatinase activity was increased in LPP-KO but not WT spheroids. The LPP-KO spheroids adhering to the collagen gel responded with decreased contraction to potassium chloride. The relaxation response to caffeine and norepinephrine was also smaller in the LPP-KO spheroids than in their WT counterparts. The overexpression of zyxin in LPP-KO VSMCs resulted in a reversal to a more quiescent differentiated phenotype. In native VSMCs, i.e., in isolated perfused segments of the mesenteric artery (MA), the contractile responses of LPP-KO segments to potassium chloride, phenylephrine or endothelin-1 did not vary from those in isolated perfused WT segments. In contrast, the myogenic response of LPP-KO MA segments was significantly attenuated while zyxin-deficient MA segments displayed a normal myogenic response. We propose that LPP, which we found to be expressed solely in the medial layer of different arteries from adult mice, may play an important role in controlling the quiescent contractile phenotype of VSMCs

Methods for identification and confirmation of targeted subgroups in clinical trials: A systematic review.

Important objectives in the development of stratified medicines include the identification and confirmation of subgroups of patients with a beneficial treatment effect and a positive benefit-risk balance. We report the results of a literature review on methodological approaches to the design and analysis of clinical trials investigating a potential heterogeneity of treatment effects across subgroups. The identified approaches are classified based on certain characteristics of the proposed trial designs and analysis methods. We distinguish between exploratory and confirmatory subgroup analysis, frequentist, Bayesian and decision-theoretic approaches and, last, fixed-sample, group-sequential, and adaptive designs and illustrate the available trial designs and analysis strategies with published case studies.peerReviewe