Reproducibility of next-generation-sequencing-based analysis of a CRISPR/Cas9 genome edited oil seed rape.

Next-generation-sequencing (NGS) becomes increasingly important for laboratories tasked with the detection of genetically modified organisms (GMOs) in food, feed and seeds. Its implementation into standardized workflows demands reliable intra- and inter-laboratory reproducibility. Here, we analyze the reproducibility of short- and long-read targeted NGS and long-read whole genome sequencing (WGS) data between three independent laboratories. Replicate samples were submitted for sequencing and comparatively analyzed. The targeted-NGS-samples consisted of oil seed rape (OSR) sampled from a commodity shipment spiked with a genome edited (GE) OSR and the WGS-samples consisted of leaf material from the GMOs' parental line. All laboratories delivered highly reproducible high-quality targeted NGS data with little variation. The detection of GMO-related sequences works well regardless of the facility, while the mapping to the complex genome is superior using long read data. Long read WGS is currently not suitable for routine use in enforcement laboratories, due to a large inter-laboratory variation.

A Novel Anthropomorphic Phantom Composed of Tissue-Equivalent Materials for Use in Experimental Radiotherapy: Design, Dosimetry and Biological Pilot Study.

The production of anthropomorphic phantoms generated from tissue-equivalent materials is challenging but offers an excellent copy of the typical environment encountered in typical patients. High-quality dosimetry measurements and the correlation of the measured dose with the biological effects elicited by it are a prerequisite in preparation of clinical trials with novel radiotherapy approaches. We designed and produced a partial upper arm phantom from tissue-equivalent materials for use in experimental high-dose-rate radiotherapy. The phantom was compared to original patient data using density values and Hounsfield units obtained from CT scans. Dose simulations were conducted for broad-beam irradiation and microbeam radiotherapy (MRT) and compared to values measured in a synchrotron radiation experiment. Finally, we validated the phantom in a pilot experiment with human primary melanoma cells.

The Microbeam Insert at the White Beam Beamline P61A at the Synchrotron PETRA III/DESY: A New Tool for High Dose Rate Irradiation Research.

High dose rate radiotherapies such as FLASH and microbeam radiotherapy (MRT) both have developed to the stage of first veterinary studies within the last decade. With the development of a new research tool for high dose rate radiotherapy at the end station P61A of the synchrotron beamline P61 on the DESY campus in Hamburg, we increased the research capacity in this field to speed up the translation of the radiotherapy techniques which are still experimental, from bench to bedside. At P61, dose rates of several hundred Gy/s can be delivered. Compared to dedicated biomedical beamlines, the beam width available for MRT experiments is a very restrictive factor. We developed two model systems specifically to suit these specific technical parameters and tested them in a first set of experiments.

Evaluating the Suitability of 3D Bioprinted Samples for Experimental Radiotherapy: A Pilot Study.

Radiotherapy is an important component in the treatment of lung cancer, one of the most common cancers worldwide, frequently resulting in death within only a few years of diagnosis. In order to evaluate new therapeutic approaches and compare their efficiency with regard to tumour control at a pre-clinical stage, it is important to develop standardized samples which can serve as inter-institutional outcome controls, independent of differences in local technical parameters or specific techniques. Recent developments in 3D bioprinting techniques could provide a sophisticated solution to this challenge. We have conducted a pilot project to evaluate the suitability of standardized samples generated from 3D printed human lung cancer cells in radiotherapy studies. The samples were irradiated at high dose rates using both broad beam and microbeam techniques. We found the 3D printed constructs to be sufficiently mechanically stable for use in microbeam studies with peak doses up to 400 Gy to test for cytotoxicity, DNA damage, and cancer cell death in vitro. The results of this study show how 3D structures generated from human lung cancer cells in an additive printing process can be used to study the effects of radiotherapy in a standardized manner.

Modification of the Langendorff system of the isolated beating heart for experimental radiotherapy at a synchrotron: 4000†Gy in a heart beat.

Microbeam radiotherapy could help to cure malignant tumours which are currently still considered therapy-resistant. With an irradiation target in the thoracic cavity, the heart would be one of the most important organs at risk. To assess the acute adverse effects of microbeam irradiation in the heart, a powerful ex vivo tool was created by combining the Langendorff model of the isolated beating mammalian heart with X-Tream dosimetry. In a first pilot experiment conducted at the Biomedical and Imaging Beamline of the Australian Synchrotron, the system was tested at a microbeam peak dose approximately ten times higher than the anticipated future microbeam irradiation treatment doses. The entire heart was irradiated with a dose of 4000 Gy at a dose rate of >6000 Gy s-1, using an array of 50 µm-wide microbeams spaced at a centre-to-centre distance of 400 µm. Although temporary arrhythmias were seen, they reverted spontaneously to a stable rhythm and no cardiac arrest occurred. This amazing preservation of cardiac function is promising for future therapeutic approaches.

Microbeam Irradiation of the Beating Rodent Heart: An Ex Vivo Study of Acute and Subacute Effects on Cardiac Function.

PURPOSE: Microbeam radiation therapy (MRT) has shown several advantages compared with conventional broad-beam radiation therapy in small animal models, including a better preservation of normal tissue function and improved drug delivery based on a rapidly increased vascular permeability in the target region. Normal tissue tolerance is the limiting factor in clinical radiation therapy. Knowledge of the normal tissue tolerance of organs at risk is therefore a prerequisite in evaluating any new radiation therapy approach. With an irradiation target in the thoracic cavity, the heart would be the most important organ at risk. METHODS AND MATERIALS: We used the ex vivo beating rodent heart in the Langendorff perfusion system at the synchrotron to administer microbeam irradiation (MBI) with a peak dose of 40 or 400 Gy. By continuously recording the electrocardiogram, the left ventricular pressure, and the aortic pressure before, during and after MBI, we were able to assess acute and subacute effects of MBI on electrophysiological and mechanical cardiac function. In addition, we analyzed histologic and ultrastructural sequelae caused by MBI. RESULTS: There were no significant changes in heart rate, heart rate variability, systolic increase of left ventricular pressure or aortic pressure. Moreover, the changes of heart rate, left ventricular pressure and aortic pressure by adding 10-5 mol/L norepinephrine to the perfusate, were also not significant between MBI and sham experiments. However, the rate-pressure product as a surrogate marker for maximum workload after MBI was significantly lower compared with sham-irradiated controls. On the structural level, no severe membranous, sarcomeric, mitochondrial or nuclear changes caused by MBI were detected by desmin immunohistochemistry and electron microscopy. CONCLUSIONS: With respect to acute and subacute toxicity, an MBI peak dose up to 400 Gy did not result in severe changes in cardiac electrophysiology or mechanics.

Clinical Phenotype and Management of Severe Neurotoxicity Observed in Patients with Neuroblastoma Treated with Dinutuximab Beta in Clinical Trials.

Neurotoxicity is an off-tumour, on-target side effect of GD2-directed immunotherapy with monoclonal antibodies. Here, we report the frequency, management and outcome of patients enrolled in two prospective clinical trials who experienced severe neurotoxicity during immunotherapy with the anti-GD2 antibody dinutuximab beta (DB) administered as short-term infusion (HR-NBL1/SIOPEN study, randomisation R2, EudraCT 2006-001489-17) or as long-term infusion (HR-NBL1/SIOPEN study, randomisation R4, EudraCT 2006-001489-17 and LTI/SIOPEN study, EudraCT 2009-018077-31), either alone or with subcutaneous interleukin-2 (scIL-2). The total number of patients included in this analysis was 1102. Overall, 44/1102 patients (4.0%) experienced Grade 3/4 neurotoxicities (HR-NBL1 R2, 21/406; HR-NBL1 R4, 8/408; LTI study, 15/288), including 27 patients with severe neurotoxicities (2.5%). Events occurred predominantly in patients receiving combined treatment with DB and scIL-2. Neurotoxicity was treated using dexamethasone, prednisolone, intravenous immunoglobulins and, in two patients, plasmapheresis, which was highly effective. While neurological recovery was observed in 16 of 21 patients with severe neurotoxicities, 5/1102 (0.45%) patients experienced persistent and severe neurological deficits. In conclusion, severe neurotoxicity is most commonly observed in patients receiving DB with scIL-2. Considering the lack of clinical benefit for IL-2 in clinical trials so far, the administration of IL-2 alongside DB is not recommended.

Effects of Microbeam Irradiation on Rodent Esophageal Smooth Muscle Contraction.

BACKGROUND: High-dose-rate radiotherapy has shown promising results with respect to normal tissue preservation. We developed an ex vivo model to study the physiological effects of experimental radiotherapy in the rodent esophageal smooth muscle. METHODS: We assessed the physiological parameters of the esophageal function in ex vivo preparations of the proximal, middle, and distal segments in the organ bath. High-dose-rate synchrotron irradiation was conducted using both the microbeam irradiation (MBI) technique with peak doses greater than 200 Gy and broadbeam irradiation (BBI) with doses ranging between 3.5-4 Gy. RESULTS: Neither MBI nor BBI affected the function of the contractile apparatus. While peak latency and maximal force change were not affected in the BBI group, and no changes were seen in the proximal esophagus segments after MBI, a significant increase in peak latency and a decrease in maximal force change was observed in the middle and distal esophageal segments. CONCLUSION: No severe changes in physiological parameters of esophageal contraction were determined after high-dose-rate radiotherapy in our model, but our results indicate a delayed esophageal function. From the clinical perspective, the observed increase in peak latency and decreased maximal force change may indicate delayed esophageal transit.

Perspectives for microbeam irradiation at the SYRMEP beamline.

It has been shown previously both in vitro and in vivo that microbeam irradiation (MBI) can control malignant tumour cells more effectively than the clinically established concepts of broad beam irradiation. With the aim to extend the international capacity for microbeam research, the first MBI experiment at the biomedical beamline SYRMEP of the Italian synchrotron facility ELETTRA has been conducted. Using a multislit collimator produced by the company TECOMET, arrays of quasi-parallel microbeams were successfully generated with a beam width of 50 µm and a centre-to-centre distance of 400 µm. Murine melanoma cell cultures were irradiated with a target dose of approximately 65 Gy at a mean photon energy of ∼30 keV with a dose rate of 70 Gy s-1 and a peak-to-valley dose of ∼123. This work demonstrated a melanoma cell reduction of approximately 80% after MBI. It is suggested that, while a high energy is essential to achieve high dose rates in order to deposit high treatment doses in a short time in a deep-seated target, for in vitro studies and for the treatment of superficial tumours a spectrum in the lower energy range might be equally suitable or even advantageous.

Long-Term Outcome and Role of Biology within Risk-Adapted Treatment Strategies: The Austrian Neuroblastoma Trial A-NB94.

We evaluated long-term outcome and genomic profiles in the Austrian Neuroblastoma Trial A-NB94 which applied a risk-adapted strategy of treatment (RAST) using stage, age and MYCN amplification (MNA) status for stratification. RAST ranged from surgery only to intensity-adjusted chemotherapy, single or multiple courses of high-dose chemotherapy (HDT) followed by autologous stem cell rescue depending on response to induction chemotherapy, and irradiation to the primary tumor site. Segmental chromosomal alterations (SCAs) were investigated retrospectively using multi- and pan-genomic techniques. The A-NB94 trial enrolled 163 patients. Patients with localized disease had an excellent ten-year (10y) event free survival (EFS) and overall survival (OS) of 99 ± 1% and 93 ± 2% whilst it was 80 ± 13% and 90 ± 9% for infants with stage 4S and for infants with stage 4 non-MNA disease both 83 ± 15%. Stage 4 patients either >12 months or ≤12 months but with MNA had a 10y-EFS and OS of 45 ± 8% and 47 ± 8%, respectively. SCAs were present in increasing frequencies according to stage and age: in 29% of localized tumors but in 92% of stage 4 tumors (p < 0.001), and in 39% of patients ≤ 12 months but in 63% of patients > 12 months (p < 0.001). RAST successfully reduced chemotherapy exposure in low- and intermediate-risk patients with excellent long-term results while the outcome of high-risk disease met contemporary trials.

A Mouse Model for Microbeam Radiation Therapy of the Lung.

PURPOSE: Radiation therapy is an important treatment component for patients with lung cancer. However, the survival time gained with clinical radiation therapy techniques is relatively short. Data from preclinical experiments suggest that synchrotron microbeam radiation therapy could be much better suited to control malignant brain tumors than current clinical concepts of radiation therapy. Even at peak doses of several hundred gray, the extent of functional deficits is low. METHODS AND MATERIALS: We have developed the first mouse model to study the effects of microbeam irradiation in lung tissue. RESULTS: Up to peak doses of 400 Gy, no acute adverse effects were seen. CONCLUSION: This model is well suited to explore the potential of microbeam radiation therapy in the treatment of lung cancer and the response of normal lung tissue and organs at risk.

Improved testing of recent HIV-1 infections with the BioRad avidity assay compared to the limiting antigen avidity assay and BED Capture enzyme immunoassay: evaluation using reference sample panels from the German Seroconverter Cohort.

BACKGROUND: The variety and limitations of current laboratory methods for estimating HIV-incidence has driven attempts to improve and standardize the performance of serological 'Tests for Recent HIV-Infections' (TRI). Primary and follow-up HIV-1 positive plasma samples from individuals with well-defined dates of infection collected as part of the German Seroconverter Cohort provided specimens highly suitable for use in comparing the performance of three TRIs: the AWARE™ BED™ EIA HIV-1 Incidence test (BED-CEIA), Genetic systems HIV-1/HIV-2 Plus O EIA antibody avidity-based assay (BioRad Avidity) and Sedia™ HIV-1 LAg Avidity EIA (LAg Avidity). METHODS: The evaluation panel included 180 specimens: 44 from antiretroviral (ARV)-naïve individuals with recently acquired HIV-infection (≤ 130 days; 25 B and 19 non-B subtypes) and 136 from long-term (>12 months) infected individuals [101 ARV-naïve subtype B, 16 non-B subtypes, 14 ARV-treated individuals, 5 slow progressors (SLP)]. RESULTS: For long-term infected, ARV-naïve individuals the false recent rates (FRR) of both the BioRad and LAg Avidity assays were 2% (2/101 for subtype B) and 6% (1/16 for subtype 'non-B'), while the FRR of the BED-CEIA was 7% (7/101 for subtype B) and 25% (4/16 for subtype 'non-B') (all p>0.05). Misclassification of ARV-treated individuals and SLP was rare by LAg (1/14, 0/5) and BioRad Avidity assays (2/14, 1/5) but more frequent by BED-CEIA (5/14, 3/5). Among recently-infected individuals (subtype B), 60% (15/25) were correctly classified by BED-CEIA, 88% (22/25) by BioRad Avidity and significantly fewer by LAg (48%, 12/25) compared to BioRad Avidity (p = 0.005) with a higher true-recency rate among non-B infections for all assays. CONCLUSIONS: This study using well-characterized specimens demonstrated lower FRRs for both avidity methods than with the BED-CEIA. For recently infected individuals the BioRad Avidity assay was shown to give the most accurate results.

Toward high-contrast breast CT at low radiation dose.

This study was approved by the local research ethics committee, and patient informed consent was obtained. The purpose of this study was to demonstrate that high-spatial-resolution low-dose analyzer-based x-ray computed tomography (CT) can substantially improve the radiographic contrast of breast tissue in vitro when compared with that attained by using diagnostic mammography and CT. An excised human breast tumor was examined by using analyzer-based x-ray imaging with synchrotron radiation. The correspondence between analyzer-based x-ray images and diagnostic mammograms, CT images, and histopathologic findings was determined. Calcifications and fine details of soft tissue, which are at the contrast detection limit on diagnostic mammograms, are clearly visible on planar analyzer-based x-ray images. Analyzer-based x-ray CT yields high contrast from smoothly varying internal structures, such as tumorous mass lesions, corresponding to information on actual structures seen at histopathologic analysis. The mean glandular dose of 1.9 mGy in analyzer-based x-ray CT is approximately equivalent to the dose administered during single-view screening mammography. The improved visibility of mammographically indistinguishable lesions in vitro suggests that analyzer-based x-ray CT may be a valuable method in radiographic evaluation of the breast, thereby justifying further investigations.

High-resolution CT by diffraction-enhanced x-ray imaging: mapping of breast tissue samples and comparison with their histo-pathology.

The aim of this study was to introduce high-resolution computed tomography (CT) of breast tumours using the diffraction-enhanced x-ray imaging (DEI) technique and to compare results with radiological and histo-pathological examinations. X-ray CT images of tumour-bearing breast tissue samples were acquired by monochromatic synchrotron radiation (SR). Due to the narrow beam and a large sample-to-detector distance scattering is rejected in the absorption contrast images (SR-CT). Large contrast enhancement is achieved by the use of the DEI-CT method, where the effects of refraction and scatter rejection are analysed by crystal optics. Clinical mammograms and CT images were recorded as reference material for a radiological examination. Three malignant and benign samples were studied in detail. Their radiographs were compared with optical images of stained histological sections. The DEI-CT images map accurately the morphology of the samples, including collagen strands and micro-calcifications of dimensions less than 0.1 mm. Histo-pathological examination and reading of the radiographs were done independently, and the conclusions were in general agreement. High-resolution DEI-CT images show strong contrast and permit visualization of details invisible in clinical radiographs. The radiation dose may be reduced by an order of magnitude without compromising image quality, which would make possible clinical in vivo DEI-CT with future compact SR sources.

Human breast cancer in vitro: matching histo-pathology with small-angle x-ray scattering and diffraction enhanced x-ray imaging.

Twenty-eight human breast tumour specimens were studied with small-angle x-ray scattering (SAXS), and 10 of those were imaged by the diffraction enhanced x-ray imaging (DEI) technique. The sample diameter was 20 mm and the thickness 1 mm. Two examples of ductal carcinoma are illustrated by histology images, DEI, and maps of the collagen d-spacing and scattered intensity in the Porod regime, which characterize the SAXS patterns from collagen-rich regions of the samples. Histo-pathology reveals the cancer-invaded regions, and the maps of the SAXS parameters show that in these regions the scattering signal differs significantly from scattering by the surrounding tissue, indicating a degradation of the collagen structure in the invaded regions. The DEI images show the borders between collagen and adipose tissue and provide a co-ordinate system for tissue mapping by SAXS. In addition, degradation of the collagen structure in an invaded region is revealed by fading contrast of the DEI refraction image. The 28 samples include fresh, defrosted tissue and formalin-fixed tissue. The d-values with their standard deviations are given. In the fresh samples there is a systematic 0.76% increase of the d-value in the invaded regions, averaged over 11 samples. Only intra-sample comparisons are made for the formalin-fixed samples, and with a long fixation time, the difference in the d-value stabilizes at about 0.7%. The correspondence between the DEI images, the SAXS maps and the histo-pathology suggests that definitive information on tumour growth and malignancy is obtained by combining these x-ray methods.

Comparison of synchrotron radiation angiography with conventional angiography for the diagnosis of in-stent restenosis after percutaneous transluminal coronary angioplasty.

AIMS: Synchrotron radiation angiography (SRA) is a novel tool for minimally invasive coronary artery imaging. The method uses subtraction of two images produced at energies bracketing the iodine K-edge after intravenous infusion of iodinated contrast agent. We investigated the accuracy of SRA for detecting in-stent restenosis (ISR). METHODS AND RESULTS: We recruited 57 men, 4-6 months after successful PTCA. We visualized the right coronary artery (RCA) in 27 patients with 36 stented segments [12 segments with ISR>50% by quantitative coronary angiography (QCA)], and the left anterior descending artery (LAD) in 30 patients with 37 stented segments (10 ISR). SRA and QCA were performed within 2 days of each other. Two experienced observers unaware of QCA data evaluated the SRA results. Image quality was good or excellent in most patients. Global sensitivity was 64%, specificity was 95%, and positive and negative predictive values were approximately 85%. Inter-observer kappa concordance coefficient was 0.86. False negatives involved short eccentric lesions and superimposed segments, most frequently of the LAD. False positives occurred in intermediate stenoses slightly overestimated by SRA. CONCLUSION: In men, this minimally invasive approach, using small radiation doses, detects significant ISR in the RCA, but the LAD poses difficulties because of superimposition with others structures.

Quantitative comparison between two phase contrast techniques: diffraction enhanced imaging and phase propagation imaging.

Two x-ray phase contrast imaging techniques are compared in a quantitative way for future mammographic applications: diffraction enhanced imaging (DEI) and phase propagation imaging (PPI). DEI involves, downstream of the sample, an analyser crystal acting as an angular filter for x-rays refracted by the sample. PPI simply uses the propagation (Fresnel diffraction) of the monochromatic and partially coherent x-ray beam over large distances. The information given by the two techniques is assessed by theoretical simulations and compared at the level of the experimental results for different kinds of samples (phantoms and real tissues). The imaging parameters such as the energy, the angular position of the analyser crystal in the DEI case or the sample to detector distance in the PPI case were varied in order to optimize the image quality in terms of contrast, visibility and figure of merit.

High-resolution blood-brain barrier permeability and blood volume imaging using quantitative synchrotron radiation computed tomography: study on an F98 rat brain glioma.

The authors previously provided evidence of synchrotron radiation computed tomography (SRCT) efficacy for quantitative in vivo brain perfusion measurements using monochromatic X-ray beams. However, this technique was limited for small-animal studies by partial volume effects. In this paper, high-resolution absolute cerebral blood volume and blood-brain barrier permeability coefficient measurements were obtained on a rat glioma model using SRCT and a CCD camera (47x47 microm2 pixel size). This is the first report of in vivo high-resolution brain vasculature parameter assessment. The work gives interesting perspectives to quantify brain hemodynamic changes accurately in healthy and pathological small animals.

Visualisation of calcifications and thin collagen strands in human breast tumour specimens by the diffraction-enhanced imaging technique: a comparison with conventional mammography and histology.

Six excised human breast tissue specimens carrying benign and malignant tumours were examined with the diffraction-enhanced imaging technique. Diffraction-enhanced images were compared with diagnostic screen-film mammograms and the correlation with histological information of the specimens was established. The enhanced visibility of calcifications, some of which were smaller than 0.15 mm in diameter, is reported in detail. Fine details of the structures such as strands of collagen and contours between glandular and adipose tissue, which are barely visible at the contrast detection limit in the conventional absorption-based mammograms, are clearly visible in the diffraction-enhanced images. Microscopic study of the stained histopathological sections unequivocally confirms the correlation of the radiographic findings with the morphologic changes in specimens. An increased soft tissue contrast and a combination of information obtained with disparate diffraction-enhanced images provide better visibility of mammographically indistinguishable features. This kind of additional structural information of the breast tissue is required to improve assessment accuracy and earlier detection of the breast lesions. These advances in image quality make the method a very promising candidate for mammography.

Influence of scatter reduction method and monochromatic beams on image quality and dose in mammography.

In mammography, the image contrast and dose delivered to the patient are determined by the x-ray spectrum and the scatter to primary ratio S/P. Thus the quality of the mammographic procedure is highly dependent on the choice of anode and filter material and on the method used to reduce the amount of scattered radiation reaching the detector. Synchrotron radiation is a useful tool to study the effect of beam energy on the optimization of the mammographic process because it delivers a high flux of monochromatic photons. Moreover, because the beam is naturally flat collimated in one direction, a slot can be used instead of a grid for scatter reduction. We have measured the ratio S/P and the transmission factors for grids and slots for monoenergetic synchrotron radiation. In this way the effect of beam energy and scatter rejection method were separated, and their respective importance for image quality and dose analyzed. Our results show that conventional mammographic spectra are not far from optimum and that the use of a slot instead of a grid has an important effect on the optimization of the mammographic process. We propose a simple numerical model to quantify this effect.