Feasibility and quality development of biomaterials in the pretest studies of the German National Cohort.

BACKGROUND: The German National Cohort (GNC) is designed to address research questions concerning a wide range of possible causes of major chronic diseases (e.g. cancer, diabetes, infectious, allergic, neurologic and cardiovascular diseases) as well as to identify risk factors and prognostic biomarkers for early diagnosis and prevention of these diseases. The collection of biomaterials in combination with extensive information from questionnaires and medical examinations represents one of the central study components. OBJECTIVES: In two pretest studies of the German National Cohort conducted between 2011 and 2013, a range of biomaterials from a defined number of participants was collected. Ten study centres were involved in pretest 1 and 18 study centres were involved in pretest 2. Standard operation procedures (SOP) were developed and evaluated to minimize pre-analytical artefacts during biosample collection. Within the pretest studies different aspects concerning feasibility of sample collection/preparation [pretest 1 (a)] and quality control of biomarkers and proteome analyses were investigated [pretest 1 (b), (c)]. Additionally, recruitment of study participants for specific projects and examination procedures of all study centres in a defined time period according to common standards as well as transportation and decentralized storage of biological samples were tested (pretest 2). These analyses will serve as the basis for the biomaterial collection in the main study of the GNC starting in 2014. MATERIALS AND METHODS: Participants, randomly chosen from the population (n = 1000 subjects recruited at ten study sites in pretest 1) were asked to donate blood, urine, saliva and stool samples. Additionally, nasal and oropharyngeal swabs were collected at the study sites and nasal swabs were collected by the participants at home. SOPs for sample collection, preparation, storage and transportation were developed and adopted for pretest 2. In pretest 2, 18 study sites (n = 599 subjects) collected biomaterials mostly identical to pretest 1. Biomarker analyses to test the quality of the biomaterials were performed. RESULTS: In pretest 1 and 2, it was feasible to collect all biomaterials from nearly all invited participants without major problems. The mean response rate of the subjects was 95 %. As one important result we found for example that after blood draw the cellular fraction should be separated from the plasma and serum fractions during the first hour with no significant variation for up to 6 h at 4 ℃ for all analysed biomarkers. Moreover, quality control of samples using a proteomics approach showed no significant clustering of proteins according to different storage conditions. All developed SOPs were validated for use in the main study after some adaptation and modification. Additionally, electronic and paper documentation sheets were developed and tested to record time stamps, volumes, freezing times, and aliquot numbers of the collected biomaterials. DISCUSSION: The collection of the biomaterials was feasible without major problems at all participating study sites. However, the processing times were in some cases too long. To avoid pre-analytical artefacts in sample collection, appropriate standardisation among the study sites is necessary. To achieve this, blood and urine collection will have to be adapted to specific conditions of usage of liquid handling robots, which will be available at all participating study centres in the main study of the GNC. Strict compliance with the SOPs, thorough training of the staff and accurate documentation are mandatory to obtain high sample quality for later analyses. The so obtained biomaterials represent a valuable resource for research on infectious and other common complex diseases in the GNC.

A common atopy-associated variant in the Th2 cytokine locus control region impacts transcriptional regulation and alters SMAD3 and SP1 binding.

BACKGROUND: Type 2 immune responses directed by Th2 cells and characterized by the signature cytokines IL4, IL5, and IL13 play major pathogenic roles in atopic diseases. Single nucleotide polymorphisms in the human Th2 cytokine locus in particular in a locus control region within the DNA repair gene RAD50, containing several RAD50 DNase1-hypersensitive sites (RHS), have been robustly associated with atopic traits in genome-wide association studies (GWAS). Functional variants in IL13 have been intensely studied, whereas no causative variants for the IL13-independent RAD50 signal have been identified yet. This study aimed to characterize the functional impact of the atopy-associated polymorphism rs2240032 located in the human RHS7 on cis-regulatory activity and differential binding of transcription factors. METHODS: Differential transcription factor binding was analyzed by electrophoretic mobility shift assays (EMSAs) with Jurkat T-cell nuclear extracts. Identification of differentially binding factors was performed using mass spectrometry (LC-MS/MS). Reporter vector constructs carrying either the major or minor allele of rs2240032 were tested for regulating transcriptional activity in Jurkat and HeLa cells. RESULTS: The variant rs2240032 impacts transcriptional activity and allele-specific binding of SMAD3, SP1, and additional putative protein complex partners. We further demonstrate that rs2240032 is located in an RHS7 subunit which itself encompasses repressor activity and might be important for the fine-tuning of transcription regulation within this region. CONCLUSION: The human RHS7 critically contributes to the regulation of gene transcription, and the common atopy-associated polymorphism rs2240032 impacts transcriptional activity and transcription factor binding.

Modulation of Wnt and Hedgehog signaling pathways is linked to retinoic acid-induced amelioration of chronic allograft dysfunction.

Chronic renal allograft damage (CAD) is manifested by a smoldering inflammatory process that leads to transplant glomerulopathy, diffuse interstitial fibrosis and tubular atrophy with loss of tubular structures. Using a Fischer 344 (RT1lvl) to Lewis (RT1l) rat renal allograft model, transcriptomic profiling and pathway mapping, we have previously shown that dynamic dysregulation of the Wnt signaling pathways may underlie progressive CAD. Retinoic acid, an important regulator of differentiation during vertebrate embryogenesis, can moderate the damage observed in this experimental model of CAD. We show here that subsets of the Hedgehog (Hh) and canonical Wnt signaling pathways are linked to the pathophysiology of progressive fibrosis, loss of cilia in epithelia and chronic dysfunction. Oral treatment with 13cis retinoic acid (13cRA) was found to selectively ameliorate the dysregulation of the Hh and canonical Wnt pathways associated with CAD, and lead to a general preservation of cilial structures. Interplay between these pathways helps explain the therapeutic effects of retinoic acid treatment in CAD, and suggests future targets for moderating chronic fibrosing organ damage.

Comparison of different approaches for assessment of HER2 expression on protein and mRNA level: prediction of chemotherapy response in the neoadjuvant GeparTrio trial (NCT00544765).

Human epidermal growth factor receptor 2 (HER2) testing is an essential part of pathological assessment in breast cancer patients, as HER2 provides not only prognostic but also predictive information on response to targeted therapy. So far, HER2 test accuracy of immunohistochemistry/in situ-hybridization techniques is still under debate, and more reliable and robust technologies are needed. To address this issue and to evaluate the predictive value of HER2 on chemotherapy, we investigated a cohort of 278 patients from the GeparTrio trial, a prospective neoadjuvant anthracycline/taxane-based multicenter study. In the GeparTrio trial, patients were not treated with any anti-HER2 therapy, as this was not standard therapy at this time. The HER2 status was analyzed by three different approaches: local and central evaluation using immunohistochemistry combined with in situ-hybridization as well as evaluation of HER2 mRNA expression using kinetic RT-PCR from formalin-fixed, paraffin-embedded (FFPE) tissue samples using a predefined cutoff. HER2 overexpression/amplification was observed in 37.3% (91/244) and 17.9% (41/229) of the informative samples in the local and central evaluations, respectively. Positive HER2 mRNA levels were found in 19.8% (55/278). We observed a highly significant correlation between central HER2 expression and HER2 status measured by kinetic RT-PCR (r = 0.856, P < 0.0001) and an overall agreement of 95.6% (κ statistic, 0.862, CI 0.77-0.94). Further, central HER2 as well as HER2 mRNA expression were predictors for a pathological complete response after neoadjuvant anthracycline/taxane-based primary chemotherapy in a univariate binary logistic regression analysis (OR 3.29, P = 0.002; OR 2.65, P = 0.004). The predictive value could be confirmed for the central HER2 status by multivariate analysis (OR 3.04, P = 0.027). The locally assessed HER2 status was not predictive of response to chemotherapy. Our results suggest that standardized methods are preferable for evaluation of HER2 status. The kinetic RT-PCR from FFPE tissue might be an additional approach for assessment of this important prognostic and predictive parameter but has to be confirmed by other studies.

Wnt pathway regulation in chronic renal allograft damage.

The Wnt signaling pathway, linked to development, has been proposed to be recapitulated during the progressive damage associated with chronic organ failure. Chronic allograft damage following kidney transplantation is characterized by progressive fibrosis and a smoldering inflammatory infiltrate. A modified, Fischer 344 (RT1(lvl)) to Lewis (RT1(l)) rat renal allograft model that reiterates many of the major pathophysiologic processes seen in patients with chronic allograft failure was used to study the progressive disease phenotype and specific gene product expression by immunohistochemistry and transcriptomic profiling. Central components of the Tgfb, canonical Wnt and Wnt-Ca2+ signaling pathways were significantly altered with the development of chronic damage. In the canonical Wnt pathway, Wnt3, Lef1 and Tcf1 showed differential regulation. Target genes Fn1, Cd44, Mmp7 and Nos2 were upregulated and associated with the progression of renal damage. Changes in the Wnt-Ca2+ pathway were evidenced by increased expression of Wnt6, Wnt7a, protein kinase C, Cam Kinase II and Nfat transcription factors and the target gene vimentin. No evidence for alterations in the Wnt planar cell polarity (PCP) pathway was detected. Overall results suggest cross talk between the Wnt and Tgfb signaling pathways during allograft inflammatory damage and present potential targets for therapeutic intervention.