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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.

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.

Radiological exposure scenarios involving large numbers of people require a rapid and high-throughput method to identify the unexposed, and those exposed to low- and high-dose radiation. Those with high-dose exposure, e.g., >2 Gy and depending on host characteristics, may develop severe hematological acute radiation syndrome (HARS), requiring hospitalization and treatment. Previously, we identified a set of genes that discriminated these clinically relevant groups. In the current work, we examined the utility of gene expression changes to classify 1,000 split blood samples into HARS severity scores of H0, H1 and H2-4, with the latter indicating likely hospitalization. In several previous radiation dose experiments, we determined that these HARS categories corresponded, respectively, to doses of 0 Gy (unexposed), 0.5 Gy and 5 Gy. The main purpose of this work was to assess the rapidity of blood sample processing using targeted next-generation sequencing (NGS). Peripheral blood samples from two healthy donors were X-ray irradiated in vitro and incubated at 37°C for 24 h. A total of 1,000 samples were evaluated by laboratory personnel blinded to the radiation dose. Changes in gene expression of FDXR , DDB2 , POU2AF1 and WNT3 were examined with qRT-PCR as positive controls. Targeted NGS (TREX) was used on all samples for the same four genes. Agreement using both methods was almost 78%. Using NGS, all 1,000 samples were processed within 30 h. Classification of the HARS severity categories corresponding to radiation dose had an overall agreement ranging between 90-97%. Depending on the end point, either a combination of all genes or FDXR alone (H0 HARS or unexposed) provided the best classification. Using this optimized automated methodology, we assessed 100× more samples approximately three times faster compared to standard cytogenetic studies. We showed that a small set of genes, rather than a complex constellation of genes, provided robust positive (97%) and negative (97%) predictive values for HARS categories and radiation doses of 0, 0.5 and 5 Gy. The findings of this study support the potential utility of early radiation-induced gene expression changes for high-throughput biodosimetry and rapid identification of irradiated persons in need of hospitalization.

Radiofrequency electromagnetic fields (RF-EMF) are a basic requirement of modern wireless communication technology. Statutory thresholds of RF-EMF are established to limit relevant additional heat supply in human tissue. Nevertheless, to date, questions concerning nonthermal biological effects have yet to be fully addressed. New versions of microarrays (8 × 60K v2) provide a higher resolution of whole genome gene expression to display adaptive processes in cells after irradiation. In this ex vivo/ in vitro study, we irradiated peripheral blood cells from five donors with a continuous wave of 900 MHz RF-EMF for 0, 30, 60 and 90 min. Gene expression changes ( P ≤ 0.05 and ≥twofold differences above or below the room temperature control exposed samples) were evaluated with microarray analysis. The results were compared with data from room temperature + 2°C samples. Verification of microarray results was performed using bioinformatic analyses and qRT-PCR. We registered a lack of an EMF-specific gene expression response after applying the false discovery rate adjustment (FDR), using a high-stringency approach. Low-stringency analysis revealed 483 statistically significant deregulated transcripts in all RF-EMF groups relative to the room temperature exposed samples without an association with their corresponding room temperature + 2°C controls. Nevertheless, these transcripts must be regarded as statistical artefacts due to the absence of a targeted biological response, including enrichment and network analyses administered to microarray expressed gene subset profiles. Correspondingly, 14 most promising candidate transcripts examined by qRT-PCR displayed an absence of correlation with respect to the microarray results. In conclusion, these findings indicate that 900 MHz EMF exposure establishing an average specific absorption rate of 9.3 W/kg to whole blood cells is insufficient to induce nonthermal effects in gene expression during short-time exposure up to 90 min.

Purpose: This collaboration of five established European gene expression labs investigated the potential impact of culture conditions on the transcriptional response of peripheral blood to radiation exposure., Materials and Methods: Blood from one healthy donor was exposed ex vivo to a Cobalt 60 source to produce a calibration curve in addition to four unknown doses. After exposure, the blood samples were either diluted with RPMI medium or left untouched. After 24-h incubation at 37 °C the diluted blood samples were lysed, while the undiluted samples were mixed with the preservative RNALater and all samples were shipped frozen to the participating labs. Samples were processed by each lab using microarray (one lab) and QRT-PCR (four labs)., Results: We show that although culture conditions affect the total amount of RNA recovered (p < .0001) and its integrity (p < .0001), it does not significantly affect dose estimates (except for the true dose at 1.1 Gy). Most importantly, the different analysis approaches provide comparable mean absolute difference of estimated doses relative to the true doses (p = .9) and number of out of range (>0.5 Gy) measurements (p = .6)., Conclusion: This study confirms the robustness of gene expression as a method for biological dosimetry.

We implemented a two-stage study to predict late occurring hematologic acute radiation syndrome (HARS) in a baboon model based on gene expression changes measured in peripheral blood within the first two days after irradiation. Eighteen baboons were irradiated to simulate different patterns of partial-body and total-body exposure, which corresponded to an equivalent dose of 2.5 or 5 Gy. According to changes in blood cell counts the surviving baboons (n = 17) exhibited mild (H1-2, n = 4) or more severe (H2-3, n = 13) HARS. Blood samples taken before irradiation served as unexposed control (H0, n = 17). For stage I of this study, a whole genome screen (mRNA microarrays) was performed using a portion of the samples (H0, n = 5; H1-2, n = 4; H2-3, n = 5). For stage II, using the remaining samples and the more sensitive methodology, qRT-PCR, validation was performed on candidate genes that were differentially up- or down-regulated during the first two days after irradiation. Differential gene expression was defined as significant (P < 0.05) and greater than or equal to a twofold difference above a H0 classification. From approximately 20,000 genes, on average 46% appeared to be expressed. On day 1 postirradiation for H2-3, approximately 2-3 times more genes appeared up-regulated (1,418 vs. 550) or down-regulated (1,603 vs. 735) compared to H1-2. This pattern became more pronounced at day 2 while the number of differentially expressed genes decreased. The specific genes showed an enrichment of biological processes coding for immune system processes, natural killer cell activation and immune response (P = 1 × E-06 up to 9 × E-14). Based on the P values, magnitude and sustained differential gene expression over time, we selected 89 candidate genes for validation using qRT-PCR. Ultimately, 22 genes were confirmed for identification of H1-3 classifications and seven genes for identification of H2-3 classifications using qRT-PCR. For H1-3 classifications, most genes were constantly three to fivefold down-regulated relative to H0 over both days, but some genes appeared 10.3-fold (VSIG4) or even 30.7-fold up-regulated (CD177) over H0. For H2-3, some genes appeared four to sevenfold up-regulated relative to H0 (RNASE3, DAGLA, ARG2), but other genes showed a strong 14- to 33-fold down-regulation relative to H0 (WNT3, POU2AF1, CCR7). All of these genes allowed an almost completely identifiable separation among each of the HARS categories. In summary, clinically relevant HARS can be independently predicted with all 29 irradiated genes examined in the peripheral blood of baboons within the first two days postirradiation. While further studies are needed to confirm these findings, this model shows potential relevance in the prediction of clinical outcomes in exposed humans and as an aid in the prioritizing of medical treatment.

Background & Aims: Melatonin has been demonstrated to reduce liver damage in different models of stress. However, there is only limited information on the impact of this hormone on hepatic gene expression. The aim of this study was, to investigate the influence of melatonin or the melatonergic agonist ramelteon on hepatic gene expression profiles after haemorrhagic shock using a whole genome microarray analysis., Methods: Male Sprague-Dawley rats (200-300 g, n=4/group) underwent haemorrhagic shock (mean arterial pressure 35±5 mmHg). After 90 min of shock, animals were resuscitated with shed blood and Ringer's and treated with vehicle (5% dimethyl sulfoxide), melatonin or ramelteon (each 1.0 mg/kg intravenously). Sham-operated animals were treated likewise but did not undergo haemorrhage. After 2 h of reperfusion, the liver was harvested, and a whole genome microarray analysis was performed. Functional gene expression profiles were determined using the Panther® classification system; promising candidate genes were evaluated by quantitative polymerase chain reaction (PCR)., Results: Microarray and PCR data showed a good correlation (r(2)=0.84). A strong influence of melatonin on receptor mediated signal transduction was revealed using the functional gene expression profile analysis, whereas ramelteon mainly influenced transcription factors. Shock-induced upregulation of three candidate genes with relevant functions for hepatocytes (ppp1r15a, dusp5, rhoB) was significantly reduced by melatonin (p<0.05 vs. shock/vehicle), but not by ramelteon. Two genes previously known as haemorrhage-induced (il1b, s100a8) were transcriptionally repressed by both drugs., Conclusions: Melatonin and ramelteon appear to induce specific hepatic gene expression profiles after haemorrhagic shock in rats. The observed differences between both substances are likely to be attributable to a distinct mechanism of action in these agents., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: The response to traumatic brain injury (TBI) is complex and induces various biological pathways in all brain regions that contribute to bad outcomes. The cerebellar hypoxia after a frontal cortex injury may potentiate the pathophysiological impacts of TBI. Therefore, a gene expression analysis was conducted to determine the influence of hypoxia on TBIs., Material and Methods: Total RNA, including microRNAs, was isolated from the cerebellum of individuals who had died from severe frontal cortex injuries or due to natural causes of death (reference group)., Results: From a total of 19,596 genes, an average of 59.56% messenger RNAs (mRNAs) appeared expressed with 42 of them showing significant >2-fold differences of upregulated (n = 18) and downregulated (n = 24) genes. The validity of 14 candidate genes (with low p values and high fold differences or based on cited literature) was confirmed using qRT-PCR (Spearman correlation r(2) = 0.93). Only four genes appeared to be either upregulated (FOSB and IL6) or downregulated (HSD11B1 and HSPA12B). From a total of 667 microRNAs, altogether, 248 microRNAs appeared expressed with 13 of them showing significant differences in the mean gene expression. The combination of two mRNAs (HSPA12B/FOSB or IL6/HSD11B1) or two microRNAs (either miR-138/miR-744 or miR-195/miR-324-5p) completely discriminated both groups, a finding unaltered by potential confounders such as age at biosampling, survival time, and the postmortem interval., Conclusions: Cerebellar hypoxia markers are important to understand the pathophysiology of TBIs and could be used for therapeutic strategies or forensic purposes, e.g., to assess the severity of a brain injury.

This work answers the question of whether it is necessary to hybridize individual instead of pooled RNA samples on microarrays for screening gene targets suitable as diagnostic tools for radiation exposure scenarios, while at the same time meeting comparable microarray quality criteria. For developing new clinical diagnostic tools, a two-stage study design was employed in five projects. At first, pooled and not individual RNA samples were hybridized on microarrays for screening purposes. Potential gene candidates were selected based on their fold-change only. This was followed by a validation/quantification step using individual RNA samples and quantitative RT-PCR. Quality criteria from the screening approach with pooled RNA samples were compared with published data from the MicroArray Quality Control (MAQC) consortium that hybridized each reference RNA sample separately and established quality criteria for microarrays. When comparing both approaches, only insignificant differences for quality criteria such as false positives, sensitivity, specificity, and overall agreement were found. However, material, costs, and time were drastically reduced when hybridizing pooled RNA and gene targets applicable for clinical diagnostic purposes could be successfully selected. In search of new diagnostic tools for radiation exposure scenarios, the two stage study design using either pooled or individual RNA samples on microarrays shows comparable quality criteria, but the RNA pooling approach saves unique material, costs, and efforts and successfully selects gene targets that can be used for the desired diagnostic purposes.

Unlabelled: Treatment options for testis cancer depend on the histological subtype as well as on the clinical stage. An accurate staging is essential for correct treatment. The 'golden standard' for staging purposes is CT, but occult metastasis cannot be detected with this method. Currently, parameters such as primary tumour size, vessel invasion or invasion of the rete testis are used for predicting occult metastasis. Last year the association of these parameters with metastasis could not be validated in a new independent cohort. Gene expression analysis in testis cancer allowed discrimination between the different histological subtypes (seminoma and non-seminoma) as well as testis cancer and normal testis tissue. In a two-stage study design we (i) screened the whole genome (using human whole genome microarrays) for candidate genes associated with the metastatic stage in seminoma and (ii) validated and quantified gene expression of our candidate genes (real-time quantitative polymerase chain reaction) on another independent group. Gene expression measurements of two of our candidate genes (dopamine receptor D1 [DRD1] and family with sequence similarity 71, member F2 [FAM71F2]) examined in primary testis cancers made it possible to discriminate the metastasis status in seminoma. The discriminative ability of the genes exceeded the predictive significance of currently used histological/pathological parameters. Based on gene expression analysis the present study provides suggestions for improved individual decision making either in favour of early adjuvant therapy or increased surveillance., Objective: To evaluate the usefulness of gene expression profiling for predicting metastatic status in testicular seminoma at the time of first diagnosis compared with established clinical and pathological parameters., Patients and Methods: Total RNA was isolated from testicular tumours of metastasized patients (12 patients, clinical stage IIa-III), non-metastasized patients (40, clinical stage I) and adjacent 'normal' tissue (n = 36). The RNA was then converted into cDNA and real-time quantitative polymerase chain reaction was run on 94 candidate genes selected from previous work. Normalised gene expression of these genes and histological variables, e.g. tumour size and rete testis infiltration, were analysed using logistic regression analysis., Results: Expression of two genes (dopamine receptor D1 [DRD1] and family with sequence similarity 71, member F2 [FAM71F2], P = 0.005 and 0.024 in separate analysis and P = 0.004 and 0.016 when combining both genes, respectively) made it possible to significantly discriminate the metastasis status. Concordance increased from 77.9% (DRD1) and 72.3% (FAM71F2) in separate analysis and up to 87.7% when combining both genes in one model. Only primary tumour size in separate analysis (continuous or categorical with tumour size >6 cm) was significantly associated with metastasis (P = 0.039/P = 0.02), but concordance was lower (61%). When we combined tumour size with our two genes in one model there was no further statistical improvement or increased concordance., Conclusion: Based on gene expression analysis our study provides suggestions for improved individual decision making either in favour of early adjuvant therapy or increased surveillance., (© 2012 THE AUTHORS. BJU INTERNATIONAL © 2012 BJU INTERNATIONAL.)

Background: The aim of this study was the prediction of metastatic status in seminoma based on examination of the primary tumor., Methods: Total RNA was isolated from metastasized seminoma (n = 10, T1N1-2M0), non-metastasized seminoma (n = 21, T1-3N0M0), and corresponding normal tissues. Pooled RNA from 10 biopsies of each tissue type was hybridized on whole genome microarrays for screening purposes. Ninety-two selected gene candidates were quantitatively examined using real-time quantitative polymerase chain reaction (RTQ-PCR)., Results: Agreement in gene expression was 88% between the whole genome microarrays and RTQ-PCR. Metastasized seminoma showed 1,912 up-regulated and 2,179 down-regulated genes with ≥ 2-fold differences in gene expression compared non-metastasized seminoma. RTQ-PCR of selected genes showed that mean gene expression values were significantly reduced in metastasized compared with non-metastasized seminoma. The presence of metastases could be predicted based on an 85-gene expression signature by using logistic regression. Sensitivity and accuracy of the 10-fold cross-validation model were 77.8% and 84.2%, respectively., Conclusion: A logistic regression model using an 85 gene expression signature allowed identification of metastasized seminoma from the primary tumor with a sensitivity of 77.8%., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Immunoconjugates composed of the alpha-emitter (213)Bi and the monoclonal antibody d9MAb specifically target HSC45-M2 gastric cancer cells expressing mutant d9-E-cadherin. These conjugates efficiently killed tumor cells in a nude mouse peritoneal carcinomatosis model. To elucidate the molecular responses of HSC45-M2 cells to alpha-emitter irradiation, whole genome gene expression profiling was performed. For that purpose HSC45-M2 cells were incubated with lethal doses of (213)Bi-d9MAb. RNA was isolated at 6, 24 and 48 h after irradiation, transcribed into cDNA and hybridized to whole genome microarrays. Results of microarray analysis were validated using RTQ-PCR showing correspondence of approximately 90%. Following incubation with (213)Bi-d9MAb, 682-1125 genes showed upregulation and 666-1278 genes showed downregulation at one time point, each. Eight genes appeared upregulated and 12 genes downregulated throughout. Molecular functions and biological processes of differentially expressed genes were categorized according to the PANTHER database. Following (213)Bi-d9MAb irradiation also a time-dependent shift in terms of overrepresentation of biological processes was observed. Among the genes showing continuous upregulation, COL4A2, NEDD9 and C3 have not been associated with the cellular response to high LET radiation so far. The same holds true for WWP2, RFX3, HIST4H4 and JADE1 that showed continuous downregulation. According to PANTHER, three of the consistently upregulated (ITM2C, FLJ11000, MSMB) and downregulated (HCG9, GAS2L3, FLJ21439) genes, respectively, have not been associated with any biological process or molecular function so far. Thus, these findings revealed interesting new targets for selective elimination of tumor cells and new insights regarding response of tumor cells to alpha-emitter exposure.

Purpose: Gene expression profiles of seminoma were compared with nonseminoma to get insights into tumorigenesis., Materials and Methods: Eleven testicular tumor biopsies (five pure seminoma, six nonseminoma; pT1N0M0 to pT2N2M1) and biopsies from unaffected sites were analyzed once per patient using a macroarray (1,176 genes). On the same patients, six genes were validated using real-time quantitative (RTQ) polymerase chain reaction (PCR). Additionally, in a separate cohort of 19 patients, 24 genes selected from the macroarray were measured using RTQ-PCR., Results: (1) The agreement in gene expression was 94% between the two methods and two different patient cohorts. (2) Two features in gene expression were independent of the tumor entity: Most changes of gene expression occurred in five functional groups like "cell cycle" and "apoptosis." Genes within these groups were almost similarly (> 80%) up- or downregulated. (3) Nonseminoma were characterized by downregulated genes (75%), but in seminoma, upregulated genes (64%) prevailed. Furthermore, 64.4% of those genes that were differentially expressed in both tumor entities were usually upregulated in seminoma but downregulated in nonseminoma. A reverse pattern was found in 24.4% of such genes. Eleven percent of these genes showed a similar up- or downregulation in gene expression in both tumor entities., Conclusion: Seminoma in this preliminary study can be differentiated from nonseminoma due to almost opposing gene expression profiles (89% of the significantly differentially expressed genes) and are in line with the histological discrimination of both tumor entities. Underlying mechanisms and implications regarding the origin and tumor progression of both entities are discussed.