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.

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.

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.

Organ-based tube current modulation versus spectral shaping via tin prefiltration: What does bismuth breast shielding add in low-dose lung CT?

PURPOSE: Since organ-based tube current modulation (OBTCM) and tin prefiltration are limited on their own in lowering the dose of lung CT examinations, this experimental study was designed to investigate whether combinations with anterior patient shielding can increase the dose reduction potential. MATERIAL AND METHODS: Three pairs of scan protocols without/with breast shield (P1/P2: standard 120kVp, P3/P4: OBTCM at 100 kVp, P5/P6: Sn 100 kVp) were employed for radiation exposure and image quality comparisons on an anthropomorphic Alderson-Rando phantom. Equivalent doses were measured in eleven sites via thermoluminescent dosimetry and the effective dose was obtained by summation of the weighted organ doses. Dose-weighted contrast-to-noise ratios (CNRD) were calculated and four radiologists independently assessed the quality of images generated with each protocol. RESULTS: While no significant difference was determined between standard and OBTCM protocols regardless of breast shield (p ≥ 0.068), equivalent doses with spectral shaping were substantially lower (p ≤ 0.003). The highest effective dose was ascertained for standard scans (P1/P2: 7.3/6.8 mSv) with a dose reduction of 8.0 % via breast shielding. The use of a bismuth shield was more beneficial in OBTCM (P3/P4: 6.6/5.3 mSv) and spectral shaping (P5/P6: 0.7/0.6 mSv), reducing the effective dose by 19.8 % and 13.9 %, respectively. Subjective assessment favoured standard protocol P1 over tin prefiltration low-dose scans (p ≤ 0.032), however, no scan protocol entailed diagnostically insufficient image quality. CONCLUSIONS: Whereas breast shielding is particularly beneficial in combination with OBTCM, spectral shaping via tin prefiltration facilitates the most pronounced dose reduction in lung CT imaging with acceptable image quality.

Dose development in sinonasal imaging over the last decade - a retrospective patient study.

BACKGROUND: Computed tomography (CT) has become the primary imaging modality for visualization of the paranasal sinuses. In this retrospective, single center patient study the radiation dose development in the past 12 years in CT imaging of the paranasal sinuses was assessed. METHODS: The computed tomography dose index (CTDIVol) and dose length product (DLP) of a total of 1246 patients (average age: 41 ± 18 years, 361 females, 885 males) were evaluated, who received imaging of the paranasal sinuses either for chronic sinusitis diagnostic, preoperatively or posttraumatically. Scans were performed on three different CT scanners (Somatom Definition AS, Somatom Definition AS+, Somatom Force, all from Siemens Healthineers) and on one CBCT (Morita) ranging from 2010 to 2022. Reconstruction techniques were filtered back projection and three generations of iterative reconstruction (IRIS, SAFIRE, ADMIRE, all from Siemens Healthineers). Group comparisons were performed using either parametrical (ANOVA) or non-parametrical tests (Kruskal-Wallis Test), where applicable. RESULTS: Over the past 12 years, there was a 73%, 54%, and 66% CTDIVol reduction and a significant (p < 0.001) 72%, 33%, and 67% DLP reduction in assessing the paranasal sinuses for chronic sinusitis, preoperatively and posttraumatically, respectively. CONCLUSION: Technological developments in CT imaging, both hardware and software based, have led to a significant reduction in dose exposure in recent years. Particularly in imaging of the paranasal sinuses, the reduction of radiation exposure is of great interest due to the often young patient age and radiation-sensitive organs in the area of radiation exposure.

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.

Tin filter compared to low kV protocols - optimizing sinonasal imaging in computed tomography.

OBJECTIVES: Paranasal sinus imaging due to chronic inflammatory disease is one of the most common examinations in head and neck radiology with CT imaging considered the current gold standard. In this phantom study we analyzed different low dose CT protocols in terms of image quality, radiation exposure and subjective evaluation in order to establish an optimized scanning protocol. METHODS: In a phantom study, an Alderson phantom was scanned using 12 protocols between 70-120 kV and 25-200 mAs with and without tin filtration. For all datasets, iterative reconstruction was used. Data were objectively evaluated (image noise, (dose-weighted) contrast-to-noise ratio) and for subjective evaluation an online survey using a Likert scale was performed to reach a large group of clinically experienced reader (n = 62). The protocol was considered diagnostically insufficient if the median score was 4 and above and if more than 10% of raters scored 4 and above on the Likert scale. For an interreader agreement an ICC was calculated. To compare clinical value in relation to the applied dose and the objective image parameters, we calculated a figure of merit (FOM) and ranked the protocols accordingly. RESULTS: There was an overall moderate agreement between the 62 readers for the 12 examined CT protocols. In this phantom study, protocols with 100 kV with spectral shaping and 50-100 mAs obtained the best results for its combination of dose, image quality and clinical information value for diagnosing sinusitis (FOM 1st- 2nd place) with the 70 kV and 50 mAs as a good alternative as well (Sinusitis: FOM shared 2nd). For preoperative planning, where a higher dose is necessary, 100 kV with spectral shaping and 100 mAs achieved the overall best results (FOM 1st place) with 70 kV and 50 mAs ranking 4th. CONCLUSION: 100-kV protocols with spectral shaping or low kV protocols (70 kV) with a similarly low dose showed the best figure of merit for imaging sinonasal disease and preoperative planning. With modern scanner technology available, spectral shaping or low KV protocols should be used for sinusitis imaging.

Thermoluminescence Dosimetry in Abdominal CT for Urinary Stone Detection: Effective Radiation Dose Reduction With Tin Prefiltration at 100 kVp.

OBJECTIVES: Spectral shaping via tin prefiltration has gained recognition for dose saving in high-contrast imaging tasks. The aim of this phantom dosimetry study was to investigate whether the use of tin filters can also reduce the effective radiation dose in 100 kVp abdominal computed tomography (CT) compared with standard low-dose scans for suspected urolithiasis. METHODS: Using a third-generation dual-source CT scanner, 4 scan protocols each were used on a standard (P1-P4) and a modified obese Alderson-Rando phantom (P5-P8), in which 11 urinary stones of different compositions were placed. Hereby 1 scan protocol represented standard low-dose settings (P1/P5: 110 kVp/120 kVp), whereas 3 experimental protocols used low-kilovoltage spectral shaping (P2/P3/P4 and P6/P7/P8: 100 kVp with tin prefiltration). Radiation dose was recorded by thermoluminescent dosimeters at 24 measurement sites. For objective assessment of image quality, dose-weighted contrast-to-noise ratios were calculated and compared between scan protocols. Additional subjective image quality analysis was performed by 2 radiologists using equidistant 5-point scales for estimation image noise, artifacts, kidney stone detectability, and delineation of bone and soft tissue. RESULTS: Both conventional low-dose protocols without tin prefiltration were associated with the highest individual equivalent doses and the highest effective radiation dose in the experimental setup (P1: 0.29-6.43 mGy, 1.45-1.83 mSv; P5: 0.50-9.35 mGy, 2.33-2.79 mSv). With no false-positive diagnoses, both readers correctly detected each of the 11 urinary calculi irrespective of scan protocol and phantom configuration. Protocols using spectral shaping via tin prefiltration allowed for effective radiation dose reduction of up to 38% on the standard phantom and 18% on the modified obese phantom, while maintaining overall diagnostic image quality. Effective dose was approximately 10% lower in a male versus female anatomy and could be reduced by another 10% if gonadal protection was used ( P < 0.001). CONCLUSIONS: Spectral shaping via tin prefiltration at 100 kVp is a suitable means to reduce the effective radiation dose in abdominal CT imaging of patients with suspected urolithiasis. The dose reduction potential is slightly less pronounced in a modified phantom emulating an obese body composition compared with a standard phantom.

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.

Low-Dose CT Imaging of the Pelvis in Follow-up Examinations-Significant Dose Reduction and Impact of Tin Filtration: Evaluation by Phantom Studies and First Systematic Retrospective Patient Analyses.

OBJECTIVES: Low-dose (LD) computed tomography (CT) is still rarely used in musculoskeletal (MSK) radiology. This study evaluates the potentials of LD CT for follow-up pelvic imaging with special focus on tin filtration (Sn) technology for normal and obese patients with and without metal implants. MATERIALS AND METHODS: In a phantom study, 5 different LD and normal-dose (ND) CT protocols with and without tin filtration were tested using a normal and an obese phantom. Iterative reconstruction (IR) and filtered back projection (FBP) were used for CT image reconstruction. In a subsequent retrospective patient study, ND CT images of 45 patients were compared with follow-up tin-filtered LD CT images with a 90% dose reduction. Sixty-four percent of patients contained metal implants at the follow-up examination. Computed tomography images were objectively (image noise, contrast-to-noise ratio [CNR], dose-normalized contrast-to-noise ratio [CNRD]) and subjectively, using a 6-point Likert score, evaluated. In addition, the figure of merit was calculated. For group comparisons, paired t tests, Wilcoxon signed rank test, analysis of variance, or Kruskal-Wallis tests were used, where applicable. RESULTS: The LD Sn protocol with 67% dose reduction resulted in equal values in qualitative (Likert score) and quantitative image analysis (image noise) compared with the ND protocol in the phantom study. For follow-up examinations, dose could be reduced up to 90% by using Sn LD CT scans without impairment in the clinical study. However, metal implants resulted in a mild impairment of Sn LD as well as ND CT images. Cancellous bone ( P < 0.001) was assessed worse and cortical bone ( P = 0.063) equally in Sn LD CT images compared with ND CT images. Figure of merit values were significant ( P ≤ 0.02) lower and hence better in Sn LD as in ND protocols. Obese patients benefited in particular from tin filtration in LD MSK imaging in terms of image noise and CNR ( P ≤ 0.05). CONCLUSIONS: Low-dose CT scans with tin filtration allow maximum dose reduction while maintaining high image quality for certain clinical purposes, for example, follow-up examinations, especially metal implant position, material loosening, and consolidation controls. Overweight patients benefit particularly from tin filter technology. Although metal implants decrease image quality in ND as well as in Sn LD CT images, this is not a relevant limitation for assessability.

Dual-energy CT in sacral fragility fractures: defining a cut-off Hounsfield unit value for the presence of traumatic bone marrow edema in patients with osteoporosis.

BACKGROUND: Demographic change entails an increasing incidence of fragility fractures. Dual-energy CT (DECT) with virtual non-calcium (VNCa) reconstructions has been introduced as a promising diagnostic method for evaluating bone microarchitecture and marrow simultaneously. This study aims to define the most accurate cut-off value in Hounsfield units (HU) for discriminating the presence and absence of bone marrow edema (BME) in sacral fragility fractures. METHODS: Forty-six patients (40 women, 6 men; 79.7 ± 9.2 years) with suspected fragility fractures of the sacrum underwent both DECT (90 kVp / 150 kVp with tin prefiltration) and MRI. Nine regions-of-interest were placed in each sacrum on DECT-VNCa images. The resulting 414 HU measurements were stratified into "edema" (n = 80) and "no edema" groups (n = 334) based on reference BME detection in T2-weighted MRI sequences. Area under the receiver operating characteristic curve was calculated to determine the desired cut-off value and an associated conspicuity range for edema detection. RESULTS: The mean density within the "edema" group of measurements (+ 3.1 ± 8.3 HU) was substantially higher compared to the "no edema" group (-51.7 ± 21.8 HU; p < 0.010). Analysis in DECT-VNCa images suggested a cut-off value of -12.9 HU that enabled sensitivity and specificity of 100% for BME detection compared to MRI. A range of HU values between -14.0 and + 20.0 is considered indicative of BME in the sacrum. CONCLUSIONS: Quantitative analysis of DECT-VNCa with a cut-off of -12.9 HU allows for excellent diagnostic accuracy in the assessment of sacral fragility fractures with associated BME. A diagnostic "one-stop-shop" approach without additional MRI is feasible.

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).

Gene expression for biodosimetry and effect prediction purposes: promises, pitfalls and future directions - key session ConRad 2021.

PURPOSE: In a nuclear or radiological event, an early diagnostic or prognostic tool is needed to distinguish unexposed from low- and highly exposed individuals with the latter requiring early and intensive medical care. Radiation-induced gene expression (GE) changes observed within hours and days after irradiation have shown potential to serve as biomarkers for either dose reconstruction (retrospective dosimetry) or the prediction of consecutively occurring acute or chronic health effects. The advantage of GE markers lies in their capability for early (1-3 days after irradiation), high-throughput, and point-of-care (POC) diagnosis required for the prediction of the acute radiation syndrome (ARS). CONCLUSIONS: As a key session of the ConRad conference in 2021, experts from different institutions were invited to provide state-of-the-art information on a range of topics including: (1) Biodosimetry: What are the current efforts to enhance the applicability of this method to perform retrospective biodosimetry? (2) Effect prediction: Can we apply radiation-induced GE changes for prediction of acute health effects as an approach, complementary to and integrating retrospective dose estimation? (3) High-throughput and point-of-care diagnostics: What are the current developments to make the GE approach applicable as a high-throughput as well as a POC diagnostic platform? (4) Low level radiation: What is the lowest dose range where GE can be used for biodosimetry purposes? (5) Methodological considerations: Different aspects of radiation-induced GE related to more detailed analysis of exons, transcripts and next-generation sequencing (NGS) were reported.

Metal Artifact Reduction With Tin Prefiltration in Computed Tomography: A Cadaver Study for Comparison With Other Novel Techniques.

OBJECTIVES: With the aging population and thus rising numbers of orthopedic implants (OIs), metal artifacts (MAs) increasingly pose a problem for computed tomography (CT) examinations. In the study presented here, different MA reduction techniques (iterative metal artifact reduction software [iMAR], tin prefilter technique, and dual-energy CT [DECT]) were compared. MATERIALS AND METHODS: Four human cadaver pelvises with OIs were scanned on a third-generation DECT scanner using tin prefilter (Sn), dual-energy (DE), and conventional protocols. Virtual monoenergetic CT images were generated from DE data sets. Postprocessing of CT images was performed using iMAR. Qualitative (bony structures, MA, image noise) image analysis using a 6-point Likert scale and quantitative image analysis (contrast-to-noise ratio, standard deviation of background noise) were performed by 2 observers. Statistical testing was performed using Friedman test with Nemenyi test as a post hoc test. RESULTS: The iMAR Sn 150 kV protocol provided the best overall assessability of bony structures and the lowest subjective image noise. The iMAR DE protocol and virtual monochromatic image (VMI) ± iMAR achieved the most effective metal artifact reduction (MAR) (P < 0.05 compared with conventional protocols). Bony structures were rated worse in VMI ± iMAR (P < 0.05) than in tin prefilter protocols ± iMAR. The DE protocol ± iMAR had the lowest contrast-to-noise ratio (P < 0.05 compared with iMAR standard) and the highest image noise (P < 0.05 compared with iMAR VMI). The iMAR reduced MA very efficiently. CONCLUSIONS: When considering MAR and image quality, the iMAR Sn 150 kV protocol performed best overall in CT images with OI. The iMAR generated new artifacts that impaired image quality. The DECT/VMI reduced MA best, but experienced from a lack of resolution of bony fine structures.

Gene expression changes in male and female rhesus macaque 60 days after irradiation.

PURPOSE: Transcriptome changes can be expected in survivors after lethal irradiation. We aimed to characterize these in males and females and after different cytokine treatments 60 days after irradiation. MATERIAL AND METHODS: Male and female rhesus macaques (n = 142) received a whole-body exposure with 700 cGy, from which 60 animals survived. Peripheral whole blood was drawn pre-exposure and before sacrificing the surviving animals after 60 days. RESULTS: We evaluated gene expression in a three-phase study design. Phase I was a whole-genome screening (NGS) for mRNAs using five pre- and post-exposure RNA samples from both sexes (n = 20). Differential gene expression (DGE) was calculated between samples of survivors and pre-exposure samples (reference), separately for males and females. 1,243 up- and down-regulated genes were identified with 30-50% more deregulated genes in females. 37 candidate mRNAs were chosen for qRT-PCR validation in phase II using the remaining samples (n = 117). Altogether 17 genes showed (borderline) significant (t-test) DGE in groups of untreated or treated animals. Nine genes (CD248, EDAR, FAM19A5, GAL3ST4, GCNT4, HBG2/1, LRRN1, NOG, SYT14) remained with significant changes and were detected in at least 50% of samples per group. Panther analysis revealed an overlap between both sexes, related to the WNT signaling pathway, cell adhesion and immunological functions. For phase III, we validated the nine genes with candidate genes (n = 32) from an earlier conducted study on male baboons. Altogether 14 out of 41 genes showed a concordantly DGE across both species in a bilateral comparison. CONCLUSIONS: Sixty days after radiation exposure, we identified (1) sex and cytokine treatment independent transcriptional changes, (2) females with almost twice as much deregulated genes appeared more radio-responsive than males, (3) Panther analysis revealed an association with immunological processes and WNT pathway for both sexes.

Gene expression changes and DNA damage after ex vivo exposure of peripheral blood cells to various CT photon spectra.

Dual-energy CT provides enhanced diagnostic power with similar or even reduced radiation dose as compared to single-energy CT. Its principle is based on the distinct physical properties of low and high energetic photons, which, however, may also affect the biological effectiveness and hence the extent of CT-induced cellular damage. Therefore, a comparative analysis of biological effectiveness of dual- and single-energy CT scans with focus on early gene regulation and frequency of radiation-induced DNA double strand breaks (DSBs) was performed. Blood samples from three healthy individuals were irradiated ex vivo with single-energy (80 kV and 150 kV) and dual-energy tube voltages (80 kV/Sn150kV) employing a modern dual source CT scanner resulting in Volume Computed Tomography Dose Index (CTDIvol) of 15.79-18.26 mGy and dose length product (DLP) of 606.7-613.8 mGy*cm. Non-irradiated samples served as a control. Differential gene expression in peripheral blood mononuclear cells was analyzed 6 h after irradiation using whole transcriptome sequencing. DSB frequency was studied by 53BP1 + γH2AX co-immunostaining and microscopic evaluation of their focal accumulation at DSBs. Neither the analysis of gene expression nor DSB frequency provided any evidence for significantly increased biological effectiveness of dual-energy CT in comparison to samples irradiated with particular single-energy CT spectra. Relative to control, irradiated samples were characterized by a significantly higher rate of DSBs (p < 0.001) and the shared upregulation of five genes, AEN, BAX, DDB2, FDXR and EDA2R, which have already been suggested as radiation-induced biomarkers in previous studies. Despite steadily decreasing doses, CT diagnostics remain a genotoxic stressor with impact on gene regulation and DNA integrity. However, no evidence was found that varying X-ray spectra of CT impact the extent of cellular damage.

Acute radiation syndrome-related gene expression in irradiated peripheral blood cell populations.

PURPOSE: In a nuclear or radiological event, an early diagnostic tool is needed to distinguish the worried well from those individuals who may later develop life-threatenFing hematologic acute radiation syndrome. We examined the contribution of the peripheral blood's cell populations on radiation-induced gene expression (GE) changes. MATERIALS AND METHODS: EDTA-whole-blood from six healthy donors was X-irradiated with 0 and 4Gy and T-lymphocytes, B-lymphocytes, NK-cells and granulocytes were separated using immunomagnetic methods. GE were examined in cell populations and whole blood. RESULTS: The cell populations contributed to the total RNA amount with a ratio of 11.6 for T-lymphocytes, 1.2 for B-cells, 1.2 for NK-cells, 1.0 for granulocytes. To estimate the contribution of GE per cell population, the baseline (0Gy) and the radiation-induced fold-change in GE relative to unexposed was considered for each gene. The T-lymphocytes (74.8%/80.5%) contributed predominantly to the radiation-induced up-regulation observed for FDXR/DDB2 and the B-lymphocytes (97.1%/83.8%) for down-regulated POU2AF1/WNT3 with a similar effect on whole blood gene expression measurements reflecting a corresponding order of magnitude. CONCLUSIONS: T- and B-lymphocytes contributed predominantly to the radiation-induced up-regulation of FDXR/DDB2 and down-regulation of POU2AF1/WNT3. This study underlines the use of FDXR/DDB2 for biodosimetry purposes and POU2AF1/WNT3 for effect prediction of acute health effects.

Identifying a Diagnostic Window for the Use of Gene Expression Profiling to Predict Acute Radiation Syndrome.

In the event of a mass casualty radiological or nuclear scenario, it is important to distinguish between the unexposed (worried well), low-dose exposed individuals and those developing the hematological acute radiation syndrome (HARS) within the first three days postirradiation. In previous baboon studies, we identified altered gene expression changes after irradiation, which were predictive for the later developing HARS severity. Similar changes in the expression of four of these genes were observed using an in vitro human whole blood model. However, these studies have provided only limited information on the time frame of the changes after exposure in relationship to the development of HARS. In this study we analyzed the time-dependent changes in mRNA expression after in vitro irradiation of whole blood. Changes in the expression of informative mRNAs (FDXR, DBB2, POU2AF1 and WNT3) were determined in the blood of eight healthy donors (6 males, 2 females) after irradiation at 0 (control), 0.5, 2 and 4 Gy using qRT-PCR. FDXR expression was significantly upregulated (P < 0.001) 4 h after ≥0.5 Gy irradiation, with an 18-40-fold peak attained 4-12 h postirradiation which remained elevated (4-9-fold) at 72 h. DDB2 expression was upregulated after 4 h (fold change, 5-8, P < 0.001 at ≥ 0.5 Gy) and remained upregulated (3-4-fold) until 72 h (P < 0.001). The earliest time points showing a significant downregulation of POU2AF1 and WNT3 were 4 h (fold change = 0.4, P = 0.001, at 4 Gy) and 8 h (fold change = 0.3-0.5, P < 0.001, 2-4 Gy), respectively. These results indicate that the diagnostic window for detecting HARS-predictive changes in gene expression may be opened as early as 2 h for most (75%) and at 4 h postirradiation for all individuals examined. Depending on the RNA species studied this may continue for at least three days postirradiation.