Calibration curve for radiation dose estimation using FDXR gene expression biodosimetry - premises and pitfalls.

PURPOSE: Radiation-induced alterations in gene expression show great promise for dose reconstruction and for severity prediction of acute health effects. Among several genes explored as potential biomarkers, FDXR is widely used due to high upregulation in white blood cells following radiation exposure. Nonetheless, the absence of a standardized protocols for gene expression-based biodosimetry is a notable gap that warrants attention to enhance the accuracy, reproducibility and reliability. The objective of this study was to evaluate the sensitivity of transcriptional biodosimetry to differences in protocols used by different laboratories and establish guidelines for the calculation of calibration curve using FDXR expression data. MATERIAL AND METHODS: Two sets of irradiated blood samples generated during RENEB exercise were used. The first included samples irradiated with known doses including: 0, 0.25, 0.5, 1, 2, 3 and 4 Gy. The second set consisted of three 'blind' samples irradiated with 1.8 Gy, 0.4 Gy and a sham-irradiated sample. After irradiation, samples were incubated at 37 °C over 24 h and sent to participating laboratories, where RNA isolation and FDXR expression analysis by qPCR were performed using sets of primers/probes and reference genes specific for each laboratory. Calibration curves based on FDXR expression data were generated using non-linear and linear regression and used for dose estimation of 'blind' samples. RESULTS: Dose estimates for sham-irradiated sample (0.020-0.024 Gy) and sample irradiated with 0.4 Gy (0.369-0.381 Gy) showed remarkable consistency across all laboratories, closely approximating the true doses regardless variation in primers/probes and reference genes used. For sample irradiated with 1.8 Gy the dose estimates were less precise (1.198-2.011 Gy) but remained within an acceptable margin for triage within the context of high dose range. CONCLUSION: Methodological differences in reference genes and primers/probes used for FDXR expression measurement do not have a significant impact on the dose estimates generated, provided that all reference genes performed as expected and the primers/probes target a similar set of transcript variants. The preferred method for constructing a calibration curve based on FDXR expression data involves employing linear regression to establish a function that describes the relationship between the logarithm of absorbed dose and FDXR ΔCt values. However, one should be careful with using non-irradiated sample data as these cannot be accurately represented on a logarithmic scale. A standard curve generated using this approach can give reliable dose estimations in a dose range from 50 mGy to 4 Gy at least.

Liquid Biopsy in Whole Blood for Identification of Gene Expression Patterns (mRNA and miRNA) Associated with Recurrence of Glioblastoma WHO CNS Grade 4.

GBM WHO CNS Grade 4 represents a major challenge for oncology due to its aggressive behavior. Conventional imaging has restrictions in detecting tumor recurrence. This prospective study aims to identify gene-based biomarkers in whole blood instead of isolating exosomes for the early detection of tumor recurrence. Blood samples (n = 33) were collected from seven GBM patients at time points before and after surgery as well as upon tumor recurrence. Four tumor tissue samples were assessed in parallel. Next-generation sequencing (NGS), including mRNA-seq and small RNA-seq, was used to analyze gene expression profiles in blood samples and tumor tissues. A novel filtering pipeline was invented to narrow down potential candidate genes. In total, between 6-93 mRNA and 1-19 small RNA candidates could be identified among the seven patients. The overlap of genes between the patients was minimal, indicating significant inter-individual variance among GBM patients. In summary, this prospective study supports the applicability of gene expression measurements in whole blood for the detection of tumor recurrence. It might provide an alternative to the challenging workflow of liquid biopsy after laborious exosome isolation from whole blood.

Modelling the In Vivo and Ex Vivo DNA Damage Response after Internal Irradiation of Blood from Patients with Thyroid Cancer.

This work reports on a model that describes patient-specific absorbed dose-dependent DNA damage response in peripheral blood mononuclear cells of thyroid cancer patients during radioiodine therapy and compares the results with the ex vivo DNA damage response in these patients. Blood samples of 18 patients (nine time points up to 168 h post-administration) were analyzed for radiation-induced γ-H2AX + 53BP1 DNA double-strand break foci (RIF). A linear one-compartment model described the absorbed dose-dependent time course of RIF (Parameters: c characterizes DSB damage induction; k1 and k2 are rate constants describing fast and slow repair). The rate constants were compared to ex vivo repair rates. A total of 14 patient datasets could be analyzed; c ranged from 0.012 to 0.109 mGy-1, k2 from 0 to 0.04 h-1. On average, 96% of the damage is repaired quickly with k1 (range: 0.19-3.03 h-1). Two patient subgroups were distinguished by k1-values (n = 6, k1 > 1.1 h-1; n = 8, k1 < 0.6 h-1). A weak correlation with patient age was observed. While induction of RIF was similar among ex vivo and in vivo, the respective repair rates failed to correlate. The lack of correlation between in vivo and ex vivo repair rates and the applicability of the model to other therapies will be addressed in further studies.

The Influence of Computed Tomography Contrast Agent on Radiation-Induced Gene Expression and Double-Strand Breaks.

After nuclear scenarios, combined injuries of acute radiation syndrome (ARS) with, e.g., abdominal trauma, will occur and may require contrast-enhanced computed tomography (CT) scans for diagnostic purposes. Here, we investigated the effect of iodinated contrast agents on radiation-induced gene expression (GE) changes used for biodosimetry (AEN, BAX, CDKN1A, EDA2R, APOBEC3H) and for hematologic ARS severity prediction (FDXR, DDB2, WNT3, POU2AF1), and on the induction of double-strand breaks (DSBs) used for biodosimetry. Whole blood samples from 10 healthy donors (5 males, 5 females, mean age: 28 ± 2 years) were irradiated with X rays (0, 1 and 4 Gy) with and without the addition of iodinated contrast agent (0.016 ml contrast agent/ml blood) to the blood prior to the exposure. The amount of contrast agent was set to be equivalent to the blood concentration of an average patient (80 kg) during a contrast-enhanced CT scan. After irradiation, blood samples were incubated at 37°C for 20 min (DSB) and 8 h (GE, DSB). GE was measured employing quantitative real-time polymerase chain reaction. DSB foci were revealed by γH2AX + 53BP1 immunostaining and quantified automatically in >927 cells/sample. Radiation-induced differential gene expression (DGE) and DSB foci were calculated using the respective unexposed sample without supplementation of contrast agent as the reference. Neither the GE nor the number of DSB foci was significantly (P = 0.07-0.94) altered by the contrast agent application. However, for some GE and DSB comparisons with/without contrast agent, there were weakly significant differences (P = 0.03-0.04) without an inherent logic and thus are likely due to inter-individual variation. In nuclear events, the diagnostics of combined injuries can require the use of an iodinated contrast agent, which, according to our results, does not alter or influence radiation-induced GE changes and the quantity of DSB foci. Therefore, the gene expression and γH2AX focus assay can still be applied for biodosimetry and/or hematologic ARS severity prediction in such scenarios.

Validation of genes for H-ARS severity prediction in leukemia patients - interspecies comparison, challenges, and promises.

PURPOSE: In a previous baboon-study, a total of 29 genes were identified for clinical outcome prediction of the hematologic, acute, radiation, syndrome (H-ARS) severity. Among them, four genes (FDXR, DDB2, POU2AF1, WNT3) appeared promising and were validated in five leukemia patients. Within this study, we sought further in-vivo validation in a larger number of whole-body irradiated patients. MATERIAL AND METHODS: Peripheral blood was drawn from 10 leukemia patients before and up to 3 days during a fractionated (2 Gy/day) total-body irradiation (TBI) with 2-12Gy. After RNA-isolation, gene expression (GE) was evaluated on 31 genes widely used in biodosimetry and H-ARS prediction employing qRT-PCR. A customized low-density-array (LDA) allowed simultanously analyzing all genes, the 96-well format further examined the four most promising genes. Fold-changes (FC) in GE relative to pre-irradiation were calculated. RESULTS: Five patients suffering from acute-lymphoblastic-leukemia (ALL) respectively non-Hodgkin-lymphoma (NHL) revealed sufficient RNA-amounts and corresponding lymphocyte and neutrophile counts for running qRT-PCR, while acute-myeloid-leukemia (AML) and one myelofibrosis patient could not supply enough RNA. Generally, 1-2µg total RNA was isolated, whereas up to 10-fold differences in RNA-quantities (associated suppressed GE-changes) were identified among pre-exposure and exposure samples. From 31 genes, 23 were expressed in at least one of the pre-exposure samples. Relative to pre-exposure, the number of expressed genes could halve at 48 and 72h after irradiation. Using the LDA, 13 genes were validated in human samples. The four most promising genes (vid. sup.) were either undetermined or too close to pre-exposure. However, they were measured using the more sensitive 96-well format, except WNT3, which wasn´t detectable. As in previous studies, an opposite regulation in GE for FDXR in leukemia patients (up-regulated) relative to baboons (down-regulated) was reconfirmed. Radiation-induced GE-changes of DDB2 (up-regulated) and POU2AF1 (down-regulated) behaved similarly in both species. Hence, 16 out of 23 genes of two species showed GE-changes in the same direction, and up-regulated FDXR as in human studies were revalidated. CONCLUSION: Identified genes for H-ARS severity prediction, previously detected in baboons, were validated in ALL but not in AML patients. Limitations related to leukemia type, associated reduced RNA amounts, suppressed GE changes, and methodological challenges must be considered as factors negatively affecting the total number of validated genes. Based on that, we propose additional controls including blood cell counts and preferably fluorescence-based RNA quantity measurements for selecting promising samples and using a more sensitive 96-well format for candidate genes with low baseline copy numbers.

Evaluating transport conditions of conventional, widely used EDTA blood tubes for gene expression analysis in comparison to expensive, specialized PAXgene tubes in preparedness for radiological and nuclear events.

PURPOSE: Gene expression (GE) analysis of a radio-sensitive gene set (FDXR, DDB2, WNT3, POU2AF1) has been introduced in the last decade as an early and high-throughput prediction tool of later developing acute hematologic radiation syndrome (H-ARS) severity. The use of special tubes for RNA extraction from peripheral whole blood (PAXgene) represent an established standard in GE studies, although uncommonly used in clinics and not immediately available in the quantities needed in radiological/nuclear (R/N) incidents. On the other hand, EDTA blood tubes are widely utilized in clinical practice. MATERIAL AND METHODS: Using blood samples from eleven healthy donors, we investigated GE changes associated with delayed processing of EDTA tubes up to 4 h at room temperature (RT) after venipuncture (simulating delays caused by daily clinical routine), followed by a subsequent transport time of 24 h at RT, 4 °C, and -20 °C. Differential gene expression (DGE) of the target genes was further examined after X-irradiation with 0 Gy and 4 Gy under optimal transport conditions. RESULTS: No significant changes in DGE were observed when storing EDTA whole blood samples up to 4 h at RT and subsequently kept at 4 °C for 24 h which is in line with expected DGE. However, other storage conditions, such as -20 °C or RT, decreased RNA quality and/or (significantly) caused changes in DGE exceeding the known methodological variance of the qRT-PCR. CONCLUSION: Our data indicate that the use of EDTA whole blood tubes for GE-based H-ARS severity prediction is comparable to the quality of PAXgene tubes, when processed ≤ 4 h after venipuncture and the sample is transported within 24 hours at 4 °C.

Proliferation and apoptosis after whole-body irradiation: longitudinal PET study in a mouse model.

PURPOSE: A reliable method for regional in vivo imaging of radiation-induced cellular damage would be of great importance for the detection of therapy-induced injury to healthy tissue and the choice of adequate treatment of radiation emergency patients in both civilian and military events. This study aimed to investigate in a mouse model if positron emission tomography (PET) imaging with proliferation and apoptosis markers is potentially suitable for this purpose. METHODS: Four groups, including twenty mice (wild-type C57BL/6) each, were whole-body irradiated with 0 Gy, 0.5 Gy, 1 Gy, and 3 Gy and examined by PET over a six-month period at defined time points. 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) and 2-(5-[18F]fluoropentyl)-2-methyl malonic acid ([18F]ML-10) were used to visualise proliferation and apoptosis. Regional standard uptake values were compared with respect to irradiation dose over time. Histologic data and peripheral blood cell values were correlated with the PET results. RESULTS: The hematopoietic bone marrow showed a significantly increased [18F]FLT signal at early time points after radiation exposure (day 3 and day 7). This correlated with blood parameters, especially leukocytes, and histological data. A significantly increased [18F]FLT signal also occurred in the gastrointestinal tract and thymus at early time points. An increased [18F]ML-10 signal related to irradiation doses was observed in the bone marrow on day 8, but there was a high variability of standard uptake values and no correlation with histological data. CONCLUSION: [18F]FLT showed potential to visualise the extent, regional distribution and recovery from radiation-induced cellular damage in the bone marrow, gastrointestinal tract and thymus. The potential of [18F]FLT imaging to assess the extent of bone marrow affected by irradiation might be especially useful to predict the subsequent severity of hematopoietic impairment and to adapt the therapy of the bone marrow reserve. [18F]ML-10 PET proved to be not sensitive enough for the reliable detection of radiation induced apoptosis.

Nano-Architecture of Persistent Focal DNA Damage Regions in the Minipig Epidermis Weeks after Acute γ-Irradiation.

Exposure to high acute doses of ionizing radiation (IR) can induce cutaneous radiation syndrome. Weeks after such radiation insults, keratinocyte nuclei of the epidermis exhibit persisting genomic lesions that present as focal accumulations of DNA double-strand break (DSB) damage marker proteins. Knowledge about the nanostructure of these genomic lesions is scarce. Here, we compared the chromatin nano-architecture with respect to DNA damage response (DDR) factors in persistent genomic DNA damage regions and healthy chromatin in epidermis sections of two minipigs 28 days after lumbar irradiation with ~50 Gy γ-rays, using single-molecule localization microscopy (SMLM) combined with geometric and topological mathematical analyses. SMLM analysis of fluorochrome-stained paraffin sections revealed, within keratinocyte nuclei with perisitent DNA damage, the nano-arrangements of pATM, 53BP1 and Mre11 DDR proteins in γ-H2AX-positive focal chromatin areas (termed macro-foci). It was found that persistent macro-foci contained on average ~70% of 53BP1, ~23% of MRE11 and ~25% of pATM single molecule signals of a nucleus. MRE11 and pATM fluorescent tags were organized in focal nanoclusters peaking at about 40 nm diameter, while 53BP1 tags formed nanoclusters that made up super-foci of about 300 nm in size. Relative to undamaged nuclear chromatin, the enrichment of DDR protein signal tags in γ-H2AX macro-foci was on average 8.7-fold (±3) for 53BP1, 3.4-fold (±1.3) for MRE11 and 3.6-fold (±1.8) for pATM. The persistent macro-foci of minipig epidermis displayed a ~2-fold enrichment of DDR proteins, relative to DSB foci of lymphoblastoid control cells 30 min after 0.5 Gy X-ray exposure. A lasting accumulation of damage signaling and sensing molecules such as pATM and 53BP1, as well as the DSB end-processing protein MRE11 in the persistent macro-foci suggests the presence of diverse DNA damages which pose an insurmountable problem for DSB repair.

Postirradiation temperature influences DSB repair and dicentric chromosome formation-potential impact for dicentric chromosome analysis in interlaboratory comparisons.

The objective was to investigate the influence of different pre-storage temperatures in the dicentric chromosome analysis (DCA) protocol (22°C vs. 37°C) by using γ-H2AX + 53BP1 foci as a marker for deoxyribonucleic acid (DNA) double-strand break (DSB) damage induction and repair and the formation of dicentric chromosomes as a result of mis-repair. Repair of γ-H2AX + 53BP1 DSB foci was absent in samples that were incubated for 2 h at 22°C after exposure of 0.5 and 1.2 Gy. When 0.5- and 1.2-Gy-exposed samples were incubated at 37°C for 2 h, there was an average decline of 31 and 52% of DSB foci, respectively. This indicated that DNA repair occurred. There was a 27% decrease in dicentric chromosome yield at 1.2 Gy and a 15% decrease at 3.5 Gy after post-irradiation incubation for 2 h at 37°C relative to the observed dicentric frequencies at 22°C. Recommended to re-phase: our data suggested that there were more open DSBs after a 2-h incubation at 22°C, which contributed to more mis-repair and dicentric formation from the start of culture. Our findings are corroborated by publications showing that lesion interaction based on enzymatic activity is suppressed below 21°C. As such temperature variations can be a source of variation in DCA during interlaboratory comparison studies, we propose to establish a common guide for the standardisation of pre-culture conditions in cytogenetic dosimetry proficiency testing.

Effect of Expansion Media on Functional Characteristics of Bone Marrow-Derived Mesenchymal Stromal Cells.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) has been shown to rely on their immunomodulatory and regenerative properties. In order to obtain sufficient numbers of cells for clinical applications, MSCs have to be expanded ex vivo. Expansion media with xenogeneic-free (XF) growth-promoting supplements like human platelet lysate (PL) or serum- and xenogeneic-free (SF/XF) formulations have been established as safe and efficient, and both groups provide different beneficial qualities. In this study, MSCs were expanded in XF or SF/XF media as well as in mixtures thereof. MSCs cultured in these media were analyzed for phenotypic and functional properties. MSC expansion was optimal with SF/XF conditions when PL was present. Metabolic patterns, consumption of growth factors, and secretome of MSCs differed depending on the type and concentration of supplement. The lactate per glucose yield increased along with a higher proportion of PL. Many factors in the supernatant of cultured MSCs showed distinct patterns depending on the supplement (e.g., FGF-2, TGFß, and insulin only in PL-expanded MSC, and leptin, sCD40L PDGF-AA only in SF/XF-expanded MSC). This also resulted in changes in cell characteristics like migratory potential. These findings support current approaches where growth media may be utilized for priming MSCs for specific therapeutic applications.

Radiation-Induced Gene Expression Changes Used for Biodosimetry and Clinical Outcome Prediction: Challenges and Promises.

As the war in Ukraine progresses, the radiological and nuclear threat has never been as real as now. The formation of life-threatening acute radiation syndrome (ARS), in particular after the deployment of a nuclear weapon or an attack on a nuclear power station, must be considered realistic. ARS is caused by massive cell death, leading to functional organ deficits and, via systemic inflammatory responses, finally aggravates into multiple organ failure. As a deterministic effect, the severity of the disease dictates the clinical outcome. Hence, predicting ARS severity via biodosimetry or alternative approaches appears straightforward. Because the disease occurs delayed, therapy starting as early as possible has the most significant benefit. A clinically relevant diagnosis should be carried out within the diagnostic time window of about 3 days after exposure. Biodosimetry assays providing retrospective dose estimations within this time frame will support medical management decision-making. However, how closely can dose estimates be associated with the later developing ARS severity degrees when considering dose as one among other determinants of radiation exposure and cell death? From a clinical/triage point of view, ARS severity degrees can be further aggregated into unexposed, weakly diseased (no acute health effects expected), and strongly diseased patient groups, with the latter requiring hospitalization as well as an early and intensive treatment. Radiation-induced gene expression (GE) changes occur early after exposure and can be quickly quantified. GE can be used for biodosimetry purposes. Can GE be used to predict later developing ARS severity degrees and allocate individuals to the three clinically relevant groups as well?

RENEB Inter-Laboratory Comparison 2021: The Gamma-H2AX Foci Assay.

The Running the European Network of biological and retrospective dosimetry (RENEB) network of laboratories has a range of biological and physical dosimetry assays that can be deployed in the event of a radiation incident to provide exposure assessment. To maintain operational capability and provide training, RENEB runs regular inter-laboratory comparison (ILC) exercises. The RENEB ILC2021 was carried out with all the biological and physical dosimetry assays employed in the network. The focus of this paper is to evaluate the results from 6 laboratories that took part using the gamma-H2AX radiation-induced foci assay. For two laboratories this was their first RENEB ILC. Blood samples were homogenously exposed to 240 kVp X rays (1 Gy/min) to provide calibration data, (0-4 Gy), and a few weeks later three blind coded test samples, (0, 1.2 and 3.5 Gy) were prepared. All samples were allowed a 2 h repair time at 37°C before being transported, on ice packs, to the participating laboratories. On arrival, the samples were processed, scored either manually or automatically for gamma-H2AX foci and dose estimates for the 3 blind coded samples sent to the organizing laboratory. The temperature of samples during transit and the time taken to report the dose estimates were recorded. Subsequent examination of the data from each laboratory used the doses estimates to assign triage categories to the samples. After receipt of the samples, the quickest report of dose estimates was 4.6 h. Analysis of variance revealed that the laboratory carrying out the assay had a significant effect on the foci yield (P < 0.001) for the calibration data, but not on the dose estimates of the blind coded samples (P = 0.101). All laboratories correctly identified the unirradiated and irradiated samples, although the dose estimates for the latter tended to under-estimate the dose. Two participants seriously under-estimated the dose for the highly exposed sample, which resulted in the sample being placed in the lowest triage category not the highest. However, this under-estimation resulted from the samples not remaining cold during shipment, due to a delay in transit and was not related to the experience of the participating laboratory. Overall, the RENEB network laboratories have demonstrated it is possible to quickly identify a recent whole-body acute exposure using the gamma-H2AX assay within the conditions of the ILC. In addition, an ILC provides a useful training and harmonization exercise for laboratories.

International Comparison Exercise for Biological Dosimetry after Exposures with Neutrons Performed at Two Irradiation Facilities as Part of the BALANCE Project.

In the case of a radiological or nuclear event, biological dosimetry can be an important tool to support clinical decision-making. During a nuclear event, individuals might be exposed to a mixed field of neutrons and photons. The composition of the field and the neutron energy spectrum influence the degree of damage to the chromosomes. During the transatlantic BALANCE project, an exposure similar to a Hiroshima-like device at a distance of 1.5 km from the epicenter was simulated, and biological dosimetry based on dicentric chromosomes was performed to evaluate the participants ability to discover unknown doses and to test the influence of differences in neutron spectra. In a first step, calibration curves were established by irradiating blood samples with 5 doses in the range of 0-4 Gy at two different facilities in Germany (Physikalisch-Technische Bundesanstalt [PTB]) and the USA (the Columbia IND Neutron Facility [CINF]). The samples were sent to eight participating laboratories from the RENEB network and dicentric chromosomes were scored by each participant. Next, blood samples were irradiated with 4 blind doses in each of the two facilities and sent to the participants to provide dose estimates based on the established calibration curves. Manual and semiautomatic scoring of dicentric chromosomes were evaluated for their applicability to neutron exposures. Moreover, the biological effectiveness of the neutrons from the two irradiation facilities was compared. The calibration curves from samples irradiated at CINF showed a 1.4 times higher biological effectiveness compared to samples irradiated at PTB. For manual scoring of dicentric chromosomes, the doses of the test samples were mostly successfully resolved based on the calibration curves established during the project. For semiautomatic scoring, the dose estimation for the test samples was less successful. Doses >2 Gy in the calibration curves revealed nonlinear associations between dose and dispersion index of the dicentric counts, especially for manual scoring. The differences in the biological effectiveness between the irradiation facilities suggested that the neutron energy spectrum can have a strong impact on the dicentric counts.

Ex-vivo dose response characterization of the recently identified EDA2R gene after low level radiation exposures and comparison with FDXR gene expression and the γH2AX focus assay.

OBJECTIVE: Recently, promising radiation-induced EDA2R gene expression (GE) changes after low level radiation could be shown. Stimulated by that, in this study, we intended to independently validate these findings and to further characterize dose-response relationships in comparison to FDXR and the γH2AX-DNA double-strand break (DSB) focus assay, since both assays are already widely used for biodosimetry purposes. MATERIALS AND METHODS: Peripheral blood samples from six healthy human donors were irradiated ex vivo (dose: ranging from 2.6 to 49.7 mGy). Subsequently, the fold-differences relative to the sham irradiated reference group were calculated. Radiation-induced changes in GE of FDXR and EDA2R were examined using the quantitative real-time polymerase-chain-reaction (qRT-PCR). DSB foci were quantified in 100 γH2AX + 53BP1 immunostained cells employing fluorescence microscopy. Examinations were performed at single time points enabling sufficient detection of both endpoints. RESULTS: A significant increase in EDA2R GE relative to the unexposed control was observed in the range of 2.6 mGy (1.6-fold, p = .045) to 5.4 mGy (2.2-fold, p = .0002), whereas the copy numbers increased linearly up to 13.1-fold at 49.7 mGy. On the contrary, FDXR upregulation (2.2-fold) became significant after a 22.6 mGy exposure (p ≤ .02) and increased linearly up to 4-fold at 49.7 mGy. A significant increase in radiation-induced foci (relative to unexposed, RIF-fd) was observed after 11.3 mGy (RIF-fd: 1.5 ± 0.5, p ≤ .03), while the foci increased linearly up to 3-fold at 49.7 mGy. From this, the FDXR and RIF-fd slopes have shown comparability, while the EDA2R slope was five times higher. Nevertheless, the coefficient of variation (CV) of EDA2R was about 30% higher than for RIF-fd. CONCLUSION: Higher radiation-induced EDA2R GE changes and a lower radiation detection level compared to RIF-fd and FDXR GE changes examined under optimal conditions ex vivo on human samples appear promising. Yet, our results represent just the beginning of further studies to be conducted in animal models for further time- and dose-dependent evaluation and additional examinations on radiologically examined patients to evaluate the impact of confounder, such as age, sex, social behavior, or diseases.

Initial experience on abdominal photon-counting computed tomography in clinical routine: general image quality and dose exposure.

OBJECTIVES: Photon-counting computed tomography has lately found its way into clinical routine. The new technique could offer substantial improvements regarding general image quality, image noise, and radiation dose reduction. This study evaluated the first abdominal examinations in clinical routine and compared the results to conventional computed tomography. METHODS: In this single-center retrospective study, 66 patients underwent photon-counting and conventional abdominal CT. Four radiologists assessed general image quality, image noise, and image artifacts. Signal-to-noise ratio and dose properties of both techniques within the clinical application were compared. An ex vivo phantom study revealed the radiobiological impact by means of DNA double-strand break foci in peripheral blood cells by enumerating γ-H2AX+53BP1 foci. RESULTS: General image quality in accordance with the Likert scale was found superior for photon-counting CT (4.74 ± 0.46 vs. 4.25 ± 0.54; p < 0.001). Signal-to-noise ratio (p < 0.001) and also dose exposure were higher for photon-counting CT (DLP: 419.2 ± 162.2 vs. 372.3 ± 236.6 mGy*cm; p = 0.0435). CT exposure resulted in significantly increased DNA damage in comparison to sham control (p < 0.001). Investigation of the average foci per cell and radiation-induced foci numbers revealed significantly elevated numbers (p = 0.004 and p < 0.0001, respectively) after photon-counting CT. CONCLUSION: Photon-counting CT in abdominal examinations showed superior results regarding general image quality and signal-to-noise ratio in clinical routine. However, this seems to be traded for a significantly higher dose exposure and corresponding double-strand break frequency. Optimization of standard protocols in further clinical applications is required to find a compromise regarding picture quality and dose exposure. KEY POINTS: • Photon-counting computed tomography promises to enhance the diagnostic potential of medical imaging in clinical routine. • Retrospective single-center study showed superior general image quality accompanied by higher dose exposure in initial abdominal PCCT protocols compared to state-of-the-art conventional CT. • A simultaneous ex vivo phantom study revealed correspondingly increased frequencies of DNA double-strand breaks after PCCT.

Four Genes Predictive for the Severity of Hematological Damage Reveal a Similar Response after X Irradiation and Chemotherapy.

Radiological and especially nuclear accidents and incidents pose a threat to populations. In such events, gene expression (GE) analysis of a set of 4 genes (FDXR, DDB2, POU2AF1, WNT3) is an emerging approach for early and high-throughput prediction of the later manifesting severity degrees of the hematological acute radiation syndrome (H-ARS). Validation of this gene set on radiation victims is difficult since these events are rare. However, chemotherapy (CTX) is widely used e.g., breast cancer patient treatment and pathomechanisms, as well as blood cell count changes are comparable among both exposure types. We wondered whether GE changes are similarly deregulated after CTX, which would be interpreted as a confirmation of our already identified gene set for H-ARS prediction after irradiation. We examined radiation-induced differential GE (DGE) of our gene set as a positive control using in vitro whole blood samples from ten healthy donors (6 females, 4 males, aged: 24-40 years). Blood was incubated in vitro for 8 h after X irradiation with 0 and 4 Gy (1 Gy/min). These data were compared with DGE measured in vivo in blood samples of 10 breast tumor CTX patients (10 females, aged: 39-71 years) before and 4 days after administration of cyclophosphamide and epirubicin. RNA was isolated, reverse transcribed and quantitative real-time polymerase-chain-reaction (qRT-PCR) was performed to assess DGE of FDXR, DDB2, POU2AF1 and WNT3 relative to the unexposed samples using TaqMan assays. After X irradiation, we found a significant upregulation (irrespective of sex) with mean fold changes of 21 (P < 0.001) and 7 (P < 0.001) for FDXR and DDB2 and a significant down-regulation with mean fold changes of 2.5 (P < 0.001) and 2 (P = 0.005) for POU2AF1 and WNT3, respectively. After CTX, a similar pattern was observed, although mean fold changes of up-regulated FDXR (6-fold, P < 0.001) and DDB2 (3-fold, P < 0.001) as well as down-regulated POU2AF1 (1.2-fold, P = 0.270) and WNT3 (1.3-fold, P = 0.069) appeared lower corresponding to less altered blood cell count changes observed after CTX compared to historic radiation exposure data. However, a subpopulation of CTX patients (n = 6) showed on average a significant downregulation of POU2AF1 (1.8-fold, P = 0.04) and WNT3 (2.1-fold, P = 0.008). In summary, the pattern of up-regulated GE changes observed in all CTX patients and down-regulated GE changes observed in a subgroup of CTX patients appeared comparable with an already identified gene set predictive for the radiation-induced H-ARS. This underlines the significance of in vivo GE measurements in CTX patients, employed as a surrogate model to further validate already identified radiation-induced GE changes predictive for the H-ARS.

Photon-Counting Detector CT Virtual Monoengergetic Images for Cochlear Implant Visualization-A Head to Head Comparison to Energy-Integrating Detector CT.

Cochlear implants (CIs) are the primary treatment method in patients with profound sensorineural hearing loss. Interpretation of postoperative imaging with conventional energy-integrating detector computed tomography (EID-CT) following CI surgery remains challenging due to metal artifacts. Still, the photon-counting detector (PCD-CT) is a new emerging technology with the potential to eliminate these problems. This study evaluated the performance of virtual monoenergetic (VME) EID-CT images versus PCD-CT in CI imaging. In this cadaveric study, two temporal bone specimens with implanted CIs were scanned with EID-CT and PCD-CT. The images were assessed according to the visibility of interelectrode wire, size of electrode contact, and diameter of halo artifacts. The visibility of interelectrode wire sections was significantly higher when reviewing PCD-CT images. The difference in diameter measurements for electrode contacts between the two CT scanner modalities showed that the PCD-CT technology generally led to significantly larger diameter readings. The larger measurements were closer to the manufacturer's specifications for the CI electrode. The size of halo artifacts surrounding the electrode contacts did not differ significantly between the two imaging modalities. PCT-CT imaging is a promising technology for CI imaging with improved spatial resolution and better visibility of small structures than conventional EID-CT.

Identifying radiation responsive exon-regions of genes often used for biodosimetry and acute radiation syndrome prediction.

Gene expression (GE) analysis of FDXR, DDB2, WNT3 and POU2AF1 is a promising approach for identification of clinically relevant groups (unexposed, low- and high exposed) after radiological/nuclear events. However, results from international biodosimetry exercises have shown differences in dose estimates based on radiation-induced GE of the four genes. Also, differences in GE using next-generation-sequening (NGS) and validation with quantitative real-time polymerase chain reaction (qRT-PCR) was reported. These discrepancies could be caused by radiation-responsive differences among exons of the same gene. We performed GE analysis with qRT-PCR using TaqMan-assays covering all exon-regions of FDXR, DDB2, WNT3 and POU2AF1. Peripheral whole blood from three healthy donors was X-irradiated with 0, 0.5 and 4 Gy. After 24 and 48 h a dose-dependent up-regulation across almost all exon-regions for FDXR and DDB2 (4-42-fold) was found. A down-regulation for POU2AF1 (two- to threefold) and WNT3 (< sevenfold) at the 3'-end was found at 4 Gy irradiation only. Hence, this confirms our hypothesis for radiation-responsive exon-regions for WNT3 and POU2AF1, but not for FDXR and DDB2. Finally, we identified the most promising TaqMan-assays for FDXR (e.g. AR7DTG3, Hs00244586_m1), DDB2 (AR47X6H, Hs03044951_m1), WNT3 (Hs00902258_m1, Hs00902257_m1) and POU2AF1 (Hs01573370_g1, Hs01573371_m1) for biodosimetry purposes and acute radiation syndrome prediction, considering several criteria (detection limit, dose dependency, time persistency, inter-individual variability).

Repair of α-particle-induced DNA damage in peripheral blood mononuclear cells after internal ex vivo irradiation with 223Ra.

PURPOSE: As α-emitters for radiopharmaceutical therapies are administered systemically by intravenous injection, blood will be irradiated by α-particles that induce clustered DNA double-strand breaks (DSBs). Here, we investigated the induction and repair of DSB damage in peripheral blood mononuclear cells (PBMCs) as a function of the absorbed dose to the blood following internal ex vivo irradiation with [223Ra]RaCl2. METHODS: Blood samples of ten volunteers were irradiated by adding [223Ra]RaCl2 solution with different activity concentrations resulting in absorbed doses to the blood of 3 mGy, 25 mGy, 50 mGy and 100 mGy. PBMCs were isolated, divided in three parts and either fixed directly (d-samples) or after 4 h or 24 h culture. After immunostaining, the induced γ-H2AX α-tracks were counted. The time-dependent decrease in α-track frequency was described with a model assuming a repair rate R and a fraction of non-repairable damage Q. RESULTS: For 25 mGy, 50 mGy and 100 mGy, the numbers of α-tracks were significantly increased compared to baseline at all time points. Compared to the corresponding d-samples, the α-track frequency decreased significantly after 4 h and after 24 h. The repair rates R were (0.24 ± 0.05) h-1 for 25 mGy, (0.16 ± 0.04) h-1 for 50 mGy and (0.13 ± 0.02) h-1 for 100 mGy, suggesting faster repair at lower absorbed doses, while Q-values were similar. CONCLUSION: The results obtained suggest that induction and repair of the DSB damage depend on the absorbed dose to the blood. Repair rates were similar to what has been observed for irradiation with low linear energy transfer.

Public health response and medical management of internal contamination in past radiological or nuclear incidents: A narrative review.

Following a radiological or nuclear emergency, workers, responders and the public may be internally contaminated with radionuclides. Screening, monitoring and assessing any internal contamination and providing necessary medical treatment, especially when a large number of individuals are involved, is challenging. Experience gained and lessons learned from the management of previous incidents would help to identify gaps in knowledge and capabilities on preparedness for and response to radiation emergencies. In this paper, eight large-scale and five workplace radiological and nuclear incidents are reviewed cross 14 technical areas, under the broader topics of emergency preparedness, emergency response and recovery processes. The review findings suggest that 1) new strategies, algorithms and technologies are explored for rapid screening of large populations; 2) exposure assessment and dose estimation in emergency response and dose reconstruction in recovery process are supported by complementary sources of information, including 'citizen science'; 3) surge capacity for monitoring and dose assessment is coordinated through national and international laboratory networks; 4) evidence-based guidelines for medical management and follow-up of internal contamination are urgently needed; 5) mechanisms for international and regional access to medical countermeasures are investigated and implemented; 6) long-term health and medical follow up programs are designed and justified; and 7) capabilities and capacity developed for emergency response are sustained through adequate resource allocation, routine non-emergency use of technical skills in regular exercises, training, and continuous improvement.