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Inter-laboratory exercises are important tools within the European network for biological dosimetry and physical retrospective dosimetry (RENEB) to validate and improve the performance of member laboratories and to ensure an operational network with high quality standards for dose estimations in case of a large-scale radiological or nuclear event. In addition to the RENEB inter-laboratory comparison 2021, several inter-laboratory comparisons have been performed in the frame of RENEB for a number of assays in recent years. This publication gives an overview of RENEB inter-laboratory comparisons for biological dosimetry assays in the past and a final summary of the challenges and lessons learnt from the RENEB inter-laboratory comparison 2021. In addition, the dose estimates of all RENEB inter-laboratory comparisons since 2013 that have been conducted for the dicentric chromosome assay, the most established and applied assay, are compared and discussed.

The goal of the RENEB inter-laboratory comparison 2021 exercise was to simulate a large-scale radiation accident involving a network of biodosimetry labs. Labs were required to perform their analyses using different biodosimetric assays in triage mode scoring and to rapidly report estimated radiation doses to the organizing institution. This article reports the results obtained with the cytokinesis-block micronucleus assay. Three test samples were exposed to blinded doses of 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 13 mA, ∼75 keV, 1 Gy/min). These doses belong to 3 triage categories of clinical relevance: a low dose category, for no exposure or exposures inferior to 1 Gy, requiring no direct treatment of subjects; a medium dose category, with doses ranging from 1 to 2 Gy, and a high dose category, after exposure to doses higher than 2 Gy, with the two latter requiring increasing medical attention. After irradiation the test samples (no. 1, no. 2 and no. 3) were sent by the organizing laboratory to 14 centers participating in the micronucleus assay exercise. Laboratories were asked to setup micronucleus cultures and to perform the micronucleus assay in triage mode, scoring 500 binucleated cells manually, or 1,000 binucleated cells in automated/semi-automated mode. One laboratory received no blood samples, but scored pictures from another lab. Based on their calibration curves, laboratories had to provide estimates of the administered doses. The accuracy of the reported dose estimates was further analyzed by the micronucleus assay lead. The micronucleus assay allowed classification of samples in the corresponding clinical triage categories (low, medium, high dose category) in 88% of cases (manual scoring, 88%; semi-automated scoring, 100%; automated scoring, 73%). Agreement between scoring laboratories, assessed by calculating the Fleiss' kappa, was excellent (100%) for semi-automated scoring, good (83%) for manual scoring and poor (53%) for fully automated scoring. Correct classification into triage scoring dose intervals (reference dose ±0.5 Gy for doses ≤2.5 Gy, or reference dose ±1 Gy for doses >2.5 Gy), recommended for triage biodosimetry, was obtained in 79% of cases (manual scoring, 73%; semi-automated scoring, 100%; automated scoring, 67%). The percentage of dose estimates whose 95% confidence intervals included the reference dose was 58% (manual scoring, 48%; semi-automated scoring, 72%; automated scoring, 60%). For the irradiated samples no. 2 and no. 3, a systematic shift towards higher dose estimations was observed. This was also noticed with the other cytogenetic assays in this intercomparison exercise. Accuracy of the rapid triage modality could be maintained when the number of manually scored cells was scaled down to 200 binucleated cells. In conclusion, the micronucleus assay, preferably performed in a semi-automated or manual scoring mode, is a reliable technique to perform rapid biodosimetry analysis in large-scale radiation emergencies.

After large-scale radiation accidents where many individuals are suspected to be exposed to ionizing radiation, biological and physical retrospective dosimetry assays are important tools to aid clinical decision making by categorizing individuals into unexposed/minimally, moderately or highly exposed groups. Quality-controlled inter-laboratory comparisons of simulated accident scenarios are regularly performed in the frame of the European legal association RENEB (Running the European Network of Biological and Physical retrospective Dosimetry) to optimize international networking and emergency readiness in case of large-scale radiation events. In total 33 laboratories from 22 countries around the world participated in the current RENEB inter-laboratory comparison 2021 for the dicentric chromosome assay. Blood was irradiated in vitro with X rays (240 kVp, 13 mA, ∼75 keV, 1 Gy/min) to simulate an acute, homogeneous whole-body exposure. Three blood samples (no. 1: 0 Gy, no. 2: 1.2 Gy, no. 3: 3.5 Gy) were sent to each participant and the task was to culture samples, to prepare slides and to assess radiation doses based on the observed dicentric yields from 50 manually or 150 semi-automatically scored metaphases (triage mode scoring). Approximately two-thirds of the participants applied calibration curves from irradiations with γ rays and about 1/3 from irradiations with X rays with varying energies. The categorization of the samples in clinically relevant groups corresponding to individuals that were unexposed/minimally (0–1 Gy), moderately (1–2 Gy) or highly exposed (>2 Gy) was successfully performed by all participants for sample no. 1 and no. 3 and by ≥74% for sample no. 2. However, while most participants estimated a dose of exactly 0 Gy for the sham-irradiated sample, the precise dose estimates of the samples irradiated with doses >0 Gy were systematically higher than the corresponding reference doses and showed a median deviation of 0.5 Gy (sample no. 2) and 0.95 Gy (sample no. 3) for manual scoring. By converting doses estimated based on γ-ray calibration curves to X-ray doses of a comparable mean photon energy as used in this exercise, the median deviation decreased to 0.27 Gy (sample no. 2) and 0.6 Gy (sample no. 3). The main aim of biological dosimetry in the case of a large-scale event is the categorization of individuals into clinically relevant groups, to aid clinical decision making. This task was successfully performed by all participants for the 0 Gy and 3.5 Gy samples and by 74% (manual scoring) and 80% (semi-automatic scoring) for the 1.2 Gy sample. Due to the accuracy of the dicentric chromosome assay and the high number of participating laboratories, a systematic shift of the dose estimates could be revealed. Differences in radiation quality (X ray vs. γ ray) between the test samples and the applied dose effect curves can partly explain the systematic shift. There might be several additional reasons for the observed bias (e.g., donor effects, transport, experimental conditions or the irradiation setup) and the analysis of these reasons provides great opportunities for future research. The participation of laboratories from countries around the world gave the opportunity to compare the results on an international level.

Supplementary Table S1: patient characteristics including age, sex, tumor type, and treatment

Detailed information about Material and Methods used throughout the manuscript including information about cell lines, antibodies, biochemical assays and in vitro and in vivo assays.

Supplementary Figure 1-7 on irradiation of CAF; effect of irradiated CAF on cancer cells; glutamine metabolism; proteome analysis of CM CAF; effect of recombinant IGF1 on cancer cells; effect of recombinant IGF1 on metabolism of cancer cells; schematic of in vivo set-up and Figure legends or respective figures

Preoperative radiotherapy (RT) is a mainstay in the management of rectal cancer, a tumor characterized by desmoplastic stroma containing cancer-associated fibroblasts (CAF). Although CAFs are abundantly present, the effects of RT to CAF and its impact on cancer cells are unknown. We evaluated the damage responses of CAF to RT and investigated changes in colorectal cancer cell growth, transcriptome, metabolome, and kinome in response to paracrine signals emerging from irradiated CAF. RT to CAF induced DNA damage, p53 activation, cell-cycle arrest, and secretion of paracrine mediators, including insulin-like growth factor-1 (IGF1). Subsequently, RT-activated CAFs promoted survival of colorectal cancer cells, as well as a metabolic switch favoring glutamine consumption through IGF1 receptor (IGF1R) activation. RT followed by IGF1R neutralization in orthotopic colorectal cancer models reduced the number of mice with organ metastases. Activation of the downstream IGF1R mediator mTOR was significantly higher in matched (intrapatient) samples and in unmatched (interpatient) samples from rectal cancer patients after neoadjuvant chemoradiotherapy. Taken together, our data support the notion that paracrine IGF1/IGF1R signaling initiated by RT-activated CAF worsens colorectal cancer progression, establishing a preclinical rationale to target this activation loop to further improve clinical responses and patient survival.Significance: These findings reveal that paracrine IGF1/IGF1R signaling promotes colorectal cancer progression, establishing a preclinical rationale to target this activation loop. Cancer Res; 78(3); 659–70. ©2017 AACR.

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 Gy to 4 Gy at two different facilities in Germany (PTB) and USA (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 semi-automatic 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 semi-automatic scoring, the dose estimation for the test samples was less successful. Doses >2 Gy in the calibration curves revealed non-linear 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.

Tools for radiation exposure reconstruction are required to support the medical management of radiation victims in radiological or nuclear incidents. Different biological and physical dosimetry assays can be used for various exposure scenarios to estimate the dose of ionizing radiation a person has absorbed. Regular validation of the techniques through inter-laboratory comparisons (ILC) is essential to guarantee high quality results. In the current RENEB inter-laboratory comparison, the performance quality of established cytogenetic assays [dicentric chromosome assay (DCA), cytokinesis-block micronucleus assay (CBMN), stable chromosomal translocation assay (FISH) and premature chromosome condensation assay (PCC)] was tested in comparison to molecular biological assays [gamma-H2AX foci (gH2AX), gene expression (GE)] and physical dosimetry-based assays [electron paramagnetic resonance (EPR), optically or thermally stimulated luminescence (LUM)]. Three blinded coded samples (e.g., blood, enamel or mobiles) were exposed to 0, 1.2 or 3.5 Gy X-ray reference doses (240 kVp, 1 Gy/min). These doses roughly correspond to clinically relevant groups of unexposed to low exposed (0-1 Gy), moderately exposed (1-2 Gy, no severe acute health effects expected) and highly exposed individuals (>2 Gy, requiring early intensive medical care). In the frame of the current RENEB inter-laboratory comparison, samples were sent to 86 specialized teams in 46 organizations from 27 nations for dose estimation and identification of three clinically relevant groups. The time for sending early crude reports and more precise reports was documented for each laboratory and assay where possible. The quality of dose estimates was analyzed with three different levels of granularity, 1. by calculating the frequency of correctly reported clinically relevant dose categories, 2. by determining the number of dose estimates within the uncertainty intervals recommended for triage dosimetry (±0.5 Gy or ±1.0 Gy for doses 2.5 Gy), and 3. by calculating the absolute difference (AD) of estimated doses relative to the reference doses. In total, 554 dose estimates were submitted within the 6-week period given before the exercise was closed. For samples processed with the highest priority, earliest dose estimates/categories were reported within 5-10 h of receipt for GE, gH2AX, LUM, EPR, 2-3 days for DCA, CBMN and within 6-7 days for the FISH assay. For the unirradiated control sample, the categorization in the correct clinically relevant group (0-1 Gy) as well as the allocation to the triage uncertainty interval was, with the exception of a few outliers, successfully performed for all assays. For the 3.5 Gy sample the percentage of correct classifications to the clinically relevant group (≥2 Gy) was between 89-100% for all assays, with the exception of gH2AX. For the 1.2 Gy sample, an exact allocation to the clinically relevant group was more difficult and 0-50% or 0-48% of the estimates were wrongly classified into the lowest or highest dose categories, respectively. For the irradiated samples, the correct allocation to the triage uncertainty intervals varied considerably between assays for the 1.2 Gy (29-76%) and 3.5 Gy (17-100%) samples. While a systematic shift towards higher doses was observed for the cytogenetic-based assays, extreme outliers exceeding the reference doses 2-6 fold were observed for EPR, FISH and GE assays. These outliers were related to a particular material examined (tooth enamel for EPR assay, reported as kerma in enamel, but when converted into the proper quantity, i.e. to kerma in air, expected dose estimates could be recalculated in most cases), the level of experience of the teams (FISH) and methodological uncertainties (GE). This was the first RENEB ILC where everything, from blood sampling to irradiation and shipment of the samples, was organized and realized at the same institution, for several biological and physical retrospective dosimetry assays. Almost all assays appeared comparably applicable for the identification of unexposed and highly exposed individuals and the allocation of medical relevant groups, with the latter requiring medical support for the acute radiation scenario simulated in this exercise. However, extreme outliers or a systematic shift of dose estimates have been observed for some assays. Possible reasons will be discussed in the assay specific papers of this special issue. In summary, this ILC clearly demonstrates the need to conduct regular exercises to identify research needs, but also to identify technical problems and to optimize the design of future ILCs.

This two-part preclinical study aims to evaluate prostate specific membrane antigen (PSMA) as a valuable target for expression-based imaging applications and to determine changes in target binding in function of varying apparent molar activities (MAapp) of [18F]AlF-PSMA-11. For the evaluation of PSMA expression levels, male NOD/SCID mice bearing prostate cancer (PCa) xenografts of C4-2 (PSMA+++), 22Rv1 (PSMA+) and PC-3 (PSMA−) were administered [18F]AlF-PSMA-11 with a medium MAapp (20.24 ± 3.22 MBq/nmol). SUVmean and SUVmax values were respectively 3.22 and 3.17 times higher for the high versus low PSMA expressing tumors (p app, C4-2 and 22Rv1 xenograft bearing mice underwent additional [18F]AlF-PSMA-11 imaging with a high (211.2 ± 38.9 MBq/nmol) and/or low MAapp (1.92 ± 0.27 MBq/nmol). SUV values showed a significantly increasing trend with higher MAapp. Significant changes were found for SUVmean and SUVmax between the high versus low MAapp and medium versus low MAapp (both p app (p = 0.055 and 0.25, respectively). The effect of varying MAapp was more pronounced in low expressing tumors and PSMA expressing tissues (e.g. salivary glands and kidneys). Overall, administration of a high MAapp increases the detection of low expression tumors while also increasing uptake in PSMA expressing tissues, possibly leading to false positive findings. In radioligand therapy, a medium MAapp could reduce radiation exposure to dose-limiting organs with only limited effect on radionuclide accumulation in the tumor.

Gliomas, the most frequent type of primary tumor of the central nervous system in adults, results in significant morbidity and mortality. Despite the development of novel, complex, multidisciplinary, and targeted therapies, glioma therapy has not progressed much over the last decades. Therefore, there is an urgent need to develop novel patient-adjusted immunotherapies that actively stimulate antitumor T cells, generate long-term memory, and result in significant clinical benefits. This work aimed to investigate the efficacy and molecular mechanism of dendritic cell (DC) vaccines loaded with glioma cells undergoing immunogenic cell death (ICD) induced by photosens-based photodynamic therapy (PS-PDT) and to identify reliable prognostic gene signatures for predicting the overall survival of patients. Analysis of the transcriptional program of the ICD-based DC vaccine led to the identification of robust induction of Th17 signature when used as a vaccine. These DCs demonstrate retinoic acid receptor-related orphan receptor-γt dependent efficacy in an orthotopic mouse model. Moreover, comparative analysis of the transcriptome program of the ICD-based DC vaccine with transcriptome data from the TCGA-LGG dataset identified a four-gene signature (CFH, GALNT3, SMC4, VAV3) associated with overall survival of glioma patients. This model was validated on overall survival of CGGA-LGG, TCGA-GBM, and CGGA-GBM datasets to determine whether it has a similar prognostic value. To that end, the sensitivity and specificity of the prognostic model for predicting overall survival were evaluated by calculating the area under the curve of the time-dependent receiver operating characteristic curve. The values of area under the curve for TCGA-LGG, CGGA-LGG, TCGA-GBM, and CGGA-GBM for predicting five-year survival rates were, respectively, 0.75, 0.73, 0.9, and 0.69. These data open attractive prospects for improving glioma therapy by employing ICD and PS-PDT-based DC vaccines to induce Th17 immunity and to use this prognostic model to predict the overall survival of glioma patients. ispartof: CELL DEATH & DISEASE vol:13 issue:12 ispartof: location:England status: published

A wide variety of 18F-labeled PSMA-targeting PET radiotracers have been developed, including [18F]AlF-PSMA-11. As there is only limited data on the comparison with other 18F-labeled PSMA PET tracers, a comparative preclinical study between [18F]AlF-PSMA-11 and [18F]PSMA-1007 was conducted. Mice with varying PSMA expressing tumors (C4-2, 22Rv1 and PC-3, each n = 5) underwent two PET/CT scans with both [18F]AlF-PSMA-11 and [18F]PSMA-1007. Ten additional mice bearing C4-2 xenografts were subjected to ex vivo biodistribution with either [18F]AlF-PSMA-11 (n = 5) or [18F]PSMA-1007 (n = 5). Absolute SUVmean and SUVmax values were significantly higher for [18F]PSMA-1007 scans in both C4-2 tumors (p p 18F]AlF-PSMA-11 and [18F]PSMA-1007 for liver, muscle, blood and salivary glands (p > 0.05). However, in 22Rv1 xenograft bearing mice, all tumor-to-organ ratios were higher for [18F]AlF-PSMA-11 (p 18F]PSMA-1007 uptake was higher in the liver, gallbladder, small intestines and glands. Biodistribution data confirmed the increased uptake in the heart, small intestines and liver with [18F]PSMA-1007. Absolute tumor uptake was higher with [18F]PSMA-1007 in all tumors. Tumor-to-organ ratios did not differ significantly in high PSMA expressing tumors, but were higher for [18F]AlF-PSMA-11 in low PSMA expressing tumors. Furthermore, [18F]PSMA-1007 showed higher uptake in healthy organs.

A wide variety ofsup18/supF-labeled PSMA-targeting PET radiotracers have been developed, including [sup18/supF]AlF-PSMA-11. As there is only limited data on the comparison with othersup18/supF-labeled PSMA PET tracers, a comparative preclinical study between [sup18/supF]AlF-PSMA-11 and [sup18/supF]PSMA-1007 was conducted. Mice with varying PSMA expressing tumors (C4-2, 22Rv1 and PC-3, each n = 5) underwent two PET/CT scans with both [sup18/supF]AlF-PSMA-11 and [sup18/supF]PSMA-1007. Ten additional mice bearing C4-2 xenografts were subjected to ex vivo biodistribution with either [sup18/supF]AlF-PSMA-11 (n = 5) or [sup18/supF]PSMA-1007 (n = 5). Absolute SUVsubmean/suband SUVsubmax/subvalues were significantly higher for [sup18/supF]PSMA-1007 scans in both C4-2 tumors (p lt; 0.01) and 22Rv1 tumors (p lt; 0.01). In C4-2 xenograft bearing mice, the tumor-to-organ ratios did not significantly differ between [sup18/supF]AlF-PSMA-11 and [sup18/supF]PSMA-1007 for liver, muscle, blood and salivary glands (p gt; 0.05). However, in 22Rv1 xenograft bearing mice, all tumor-to-organ ratios were higher for [sup18/supF]AlF-PSMA-11 (p lt; 0.01). In healthy organs, [sup18/supF]PSMA-1007 uptake was higher in the liver, gallbladder, small intestines and glands. Biodistribution data confirmed the increased uptake in the heart, small intestines and liver with [sup18/supF]PSMA-1007. Absolute tumor uptake was higher with [sup18/supF]PSMA-1007 in all tumors. Tumor-to-organ ratios did not differ significantly in high PSMA expressing tumors, but were higher for [sup18/supF]AlF-PSMA-11 in low PSMA expressing tumors. Furthermore, [sup18/supF]PSMA-1007 showed higher uptake in healthy organs.

Even though a detailed understanding of the proliferative characteristics of T lymphocytes is imperative in many research fields, prior studies have never reached a consensus on these characteristics, and on the corresponding cell cycle kinetics specifically. In this study, the general proliferative response of human T lymphocytes to phytohaemagglutinin (PHA) stimulation was characterized using a carboxyfluorescein succinimidyl ester-based flow cytometric assay. We were able to determine when PHA-stimulated T lymphocytes complete their first division, the proportion of cells that initiate proliferation, the subsequent division rate of the cells, and the impact of irradiation on these proliferative properties. Next, we accurately visualized the cell cycle progression of dividing T lymphocytes cultured in whole blood using an adapted 5-ethynyl-2’-deoxyuridine pulse-chase method. Furthermore, through multiple downstream analysis methods, we were able to make an estimation of the corresponding cell cycle kinetics. We also visualized the impact of X-rays on the progression of the cells through the cell cycle. Our results showed dose-dependent G2 arrest after exposure to irradiation, and a corresponding delay in G1 phase-entry of the cells. In conclusion, utilizing various flow cytometric assays, we provided valuable information on T lymphocyte proliferation characteristics starting from first division to fully dividing cells.

Recent research on histologically healthy human tissues identified omnipresent mutational microclones, driven by somatic mutations known to be responsible for carcinogenesis (e.g., in TP53 or NOTCH1). These new insights are fundamentally changing current tumour evolution models, with broad oncological implications. Most studies are based on surgical remnant tissues, which are not available for many organs and rarely in a pan-organ setting (multiple organs from the same individual). Here, we describe an approach based on clinically annotated post-mortem tissues, derived from whole-body donors that are routinely used for educational purposes at human anatomy units. We validated this post-mortem approach using UV-exposed and unexposed epidermal skin tissues and confirm the presence of positively selected NOTCH1/2-, TP53- and FAT1-driven clones. No selection signals were detected in a set of immune genes or housekeeping genes. Additionally, we provide the first evidence for smoking-induced clonal changes in oral epithelia, likely underlying the origin of head and neck carcinogenesis. In conclusion, the whole-body donor-based approach provides a nearly unlimited healthy tissue resource to study mutational clonality and gain fundamental mutagenic insights in the presumed earliest stages of tumour evolution.

This two-part preclinical study aims to evaluate prostate specific membrane antigen (PSMA) as a valuable target for expression-based imaging applications and to determine changes in target binding in function of varying specific activities (SA) of 18F-AlF-PSMA-11. For the evaluation of PSMA expression levels, male NOD/SCID mice bearing prostate cancer (PCa) xenografts of C4-2 (PSMA+++), 22Rv1 (PSMA+) and PC-3 (PSMA-) were administered 18F-AlF-PSMA-11 with a medium SA (20.26 ± 3.25 MBq/µg). SUVmean and SUVmax values were respectively 3.22 and 3.17 times higher for the high vs low PSMA expressing tumors (p 18F-AlF-PSMA-11 imaging with a high (213.3 ± 39.3 MBq/µg) and/or low SA (1.94 ± 0.27 MBq/µg). SUV values showed a significantly increasing trend with higher SA. Significant changes were found for SUVmean and SUVmax between the high vs low SA and medium vs low SA (both p

Severe Combined Immune Deficiency (SCID) is a primary deficiency of the immune system in which opportunistic and recurring infections are often fatal during neonatal or infant life. SCID is caused by an increasing number of genetic defects that induce an abrogation of T lymphocyte development or function in which B and NK cells might be affected as well. Because of the increased availability and usage of next-generation sequencing (NGS), many novel variants in SCID genes are being identified and cause a heterogeneous disease spectrum. However, the molecular and functional implications of these new variants, of which some are non-coding, are often not characterized in detail. Using targeted NGS, we identified a novel homozygous c.465-1G>C splice acceptor site variant in the DCLRE1C gene in a T-B-NK+ SCID patient and fully characterized the molecular and functional impact. By performing a minigene splicing reporter assay, we revealed deregulated splicing of the DCLRE1C transcript since a cryptic splice acceptor in exon 7 was employed. This induced a frameshift and the generation of a p.Arg155Serfs*15 premature termination codon (PTC) within all DCLRE1C splice variants, resulting in the absence of full-length ARTEMIS protein. Consistently, a V(D)J recombination assay and a G0 micronucleus assay demonstrated the inability of the predicted mutant ARTEMIS protein to perform V(D)J recombination and DNA damage repair, respectively. Together, these experiments molecularly and functionally clarify how a newly identified c.465-1G>C variant in the DCLRE1C gene is responsible for inducing SCID. In a clinical context, this demonstrates how the experimental validation of new gene variants, that are identified by NGS, can facilitate the diagnosis of SCID which can be vital for implementing appropriate therapies.

Depending on the ionising radiation exposure scenario, different biological or physical assays or combinations of these assays will be required to successfully recover the scenario and to obtain reliable dose estimates to aid medical decision making for the treatment of potentially exposed individuals. Interlaboratory comparisons (ILCs) are a central tool to validate and improve the performance of methods and laboratories. In most cases, such ILCs are performed under laboratory conditions where the influence of fixed parameters contributing to the uncertainty budget can be minimized. However, in a real-life scenario, the situation is much more complicated and it is therefore crucial to validate methods and laboratory responses under more realistic conditions.

Purpose In case of a mass-casualty radiological event, there would be a need for networking to overcome surge limitations and to quickly obtain homogeneous results (reported aberration frequencies or estimated doses) among biodosimetry laboratories. These results must be consistent within such network. Inter-laboratory comparisons (ILCs) are widely accepted to achieve this homogeneity. At the European level, a great effort has been made to harmonize biological dosimetry laboratories, notably during the MULTIBIODOSE and RENEB projects. In order to continue the harmonization efforts, the RENEB consortium launched this intercomparison which is larger than the RENEB network, as it involves 38 laboratories from 21 countries. In this ILC all steps of the process were monitored, from blood shipment to dose estimation. This exercise also aimed to evaluate the statistical tools used to compare laboratory performance. Materials and methods Blood samples were irradiated at three different doses, 1.8, 0.4 and 0 Gy (samples A, C and B) with 4-MV X-rays at 0.5 Gy min-1, and sent to the participant laboratories. Each laboratory was requested to blindly analyze 500 cells per sample and to report the observed frequency of dicentric chromosomes per metaphase and the corresponding estimated dose. Results This ILC demonstrates that blood samples can be successfully distributed among laboratories worldwide to perform biological dosimetry in case of a mass casualty event. Having achieved a substantial harmonization in multiple areas among the RENEB laboratories issues were identified with the available statistical tools, which are not capable to advantageously exploit the richness of results of a large ILCs. Even though Z- and U-tests are accepted methods for biodosimetry ILCs, setting the number of analyzed metaphases to 500 and establishing a tests' common threshold for all studied doses is inappropriate for evaluating laboratory performance. Another problem highlighted by this ILC is the issue of the dose-effect curve diversity. It clearly appears that, despite the initial advantage of including the scoring specificities of each laboratory, the lack of defined criteria for assessing the robustness of each laboratory's curve is a disadvantage for the 'one curve per laboratory' model. Conclusions Based on our study, it seems relevant to develop tools better adapted to the collection and processing of results produced by the participant laboratories. We are confident that, after an initial harmonization phase reached by the RENEB laboratories, a new step toward a better optimization of the laboratory networks in biological dosimetry and associated ILC is on the way.

Purpose: Depending on the exposure scenario, different biological and/or physical assays will be required to recover the exposure situation and to obtain reliable dose estimates to aid medical decision making. Inter-laboratory comparisons (ILCs) are a central tool to validate and improve the performance of methods and laboratories. In most cases such ILCs are performed under laboratory conditions where the influence of parameters contributing to uncertainties can be minimized. In a reallife scenario, the situation is much more complicated, and it is therefore crucial to validate methods and laboratories’ abilities to carry out these methods under more realistic conditions. In 2019, EURADOS (The European Radiation Dosimetry Group) Working Group 10 (Retrospective Dosimetry) and RENEB (Running the European Network of Biological and retrospective Physical dosimetry) organized a field exercise in Lund, Sweden, to validate different methods for biological and physical retrospective dosimetry in parallel by simulating real- life exposure scenarios. Materials and methods: For the dicentric chromosome assay (DCA), 18 blood tubes were distributed across 8 thermos flasks filled with heated water and positioned at the hips and shoulders of anthropomorphic phantoms in different geometries. Two irradiations were performed with a 1.36 TBq 192Ir-source, each with two phantoms, to simulate close, distant, lateral or partially shielded exposures. For each of the blood tubes DCA dose estimates were provided by at least one laboratory and for tubes from four thermos flasks by 17 participating RENEB laboratories. In addition, blood from the different thermos flasks on each phantom was mixed to simulate heterogeneous exposures. Three radio-photoluminescence (RPL) glass dosimeters (GD) were placed at each tube to assess reference doses and the inter- and intra-tube variability. Results: The results from the DCA were homogeneous between participants and matched well with RPL GD reference doses (≥95% of estimates within ±0.5 Gy of the reference). For samples close to the source, exposed to doses >1 Gy, systematic underestimation could be corrected by accounting for exposure time. Heterogeneity within and between tubes was detected for RPL GD reference doses as well as for doses estimates from the DCA. The sample simulating a heterogeneous exposure with two different doses was detected by ~50% of the participants. Conclusions: The participants were able to successfully estimate the doses and to provide important information on the exposure scenarios under conditions closely resembling a real-life situation. The reliability of the results emphasizes the value of the DCA for retrospective dosimetry under field conditions.

OBJECTIVES The long-term outcome of tetralogy of Fallot (TOF) is determined by progressive right ventricular (RV) dysfunction through pulmonary regurgitation (PR) and the risk of malignant arrhythmia. Although mechano-electrical coupling in TOF is well-known, its time effect on the inducibility of arrhythmia remains ill-defined. The goal of this study was to investigate the mechano-electrical properties at different times in animals with chronic PR. METHODS PR was induced by a transannular patch with limited RV scarring in infant pigs. Haemodynamic assessment included biventricular pressure–volume loops after 3 (n = 8) and 6 months (n = 7) compared to controls (n = 5). The electrophysiological study included endocardial monophasic action potential registration, intraventricular conduction velocity and induction of ventricular arrhythmia by burst pacing. RESULTS Progressive RV dilation was achieved at 6 months (RV end-diastolic volume 143 ± 13 ml/m2—RV end-systolic volume 96 ± 7 ml/m2; P CONCLUSIONS In animals yielding the sequelae of a contemporary operation for TOF, mechano-electrical alterations are progressive and affect predominantly the RV after midterm exposure of PR. Because ventricular arrhythmias were not inducible despite significant RV dilation, the data suggest that the haemodynamic RV deterioration effectively precedes the risk of inducing sustained arrhythmia after TOF repair and opens a window for renewed stratification of contemporary risk factors of ventricular arrhythmias in patients operated on with currently used pulmonary valve- and RV-related techniques.

Double strand break (DSB) repair through Homologous Recombination (HR) is essential in maintaining genomic stability of the cell. Mutations in the HR pathway confer an increased risk for breast, ovarian, pancreatic and prostate cancer. PARP inhibitors (PARPi) are compounds that specifically target tumours deficient in HR. Novel PARPi are constantly being developed, but research is still heavily focussed on in vitro data, with mouse xenografts only being used in late stages of development. There is a need for assays that can: 1) provide in vivo data, 2) early in the development process of novel PARPi, 3) provide fast results and 4) at an affordable cost. Here we propose a combination of in vivo zebrafish assays to accurately quantify PARP inhibitor efficacy. We showed that PARPi display functional effects in zebrafish, generally correlating with their PARP trapping capacities. Furthermore, we displayed how olaparib mediated radiosensitization is conserved in our zebrafish model. These assays could aid the development of novel PARPi by providing early in vivo data. In addition, using zebrafish allows for high-throughput testing of combination therapies in search of novel treatment strategies.

It has previously been demonstrated that peripheral blood lymphocytes of healthy women carrying BRCA1 or BRCA2 mutations exhibit increased chromosomal radiosensitivity, which is characterized by an enhanced formation of micronuclei. These results suggest that the deficient repair of DNA double‑strand breaks may also occur in breast epithelial cells of women exhibiting a reduced expression of wild‑type BRCA1/BRCA2 proteins due to the presence of germline mutations in BRCA1/2 genes. The aim of this study was to further investigate in vitro the effects of the reduced expression of BRCA1 and BRCA2 in MCF10A human non‑tumorigenic breast epithelial cells, tentatively mimicking the phenotype of heterozygous cells of carriers of BRCA1/2 mutations. By lentivirus‑mediated RNA interference, the stable reduction of BRCA1 and BRCA2 expression at the mRNA and protein level was achieved, thus generating the BRCA1i and BRCA2i cell lines. In these cells, homologous recombination was impaired, as significantly lower yields of RAD51 foci were obtained following exposure to 2 Gy ionizing radiation compared to the control MCF10A cells (BRCA1i cells, 58% reduction; BRCA2i cells, 64% reduction). Moreover, in the BRCA1i and BRCA2i cells, a dose‑dependent increase in micronuclei was observed compared to the cells which were not subjected to gene knockdown. Cell viability was also affected by partial BRCA1/2 knockdown. On the whole, the findings of this study indicated that in cells with a reduced BRCA1 or BRCA2 expression, the impairment of homologous recombination resulted in a >50% decrease in RAD51 foci following irradiation and increased chromosomal abnormalities (micronuclei). These findings suggest that the healthy breast tissue of BRCA1/2 mutation carriers may be prone to neoplastic transformation upon exposure to diagnostic or therapeutic radiation, and that the RAD51 foci assay may be useful for the assessment of the functionality of HR repair and radiosensitivity in these women.

Age-related macular degeneration is the leading cause of blindness in the developed world. Since advanced glycation end products (AGEs) are implicated in the pathogenesis of AMD through various lines of evidence, we investigated the potential of fructosamine-3-kinase (FN3K) in the disruption of retinal AGEs, drusenoid material and drusenoid lesions in patients with AMD. AGE-type autofluorescence was measured to evaluate the effects of FN3K on glycolaldehyde-induced AGE-modified neural porcine retinas and unmodified human neural retinas. Eye pairs from cigarette-smoke- and air-exposed mice were treated and evaluated histologically. Automated optical image analysis of human tissue sections was performed to compare control- and FN3K-treated drusen and near-infrared (NIR) microspectroscopy was performed to examine biochemical differences. Optical coherence tomography (OCT) was used to evaluate the effect of FN3K on drusenoid deposits after treatment of post-mortem human eyes. FN3K treatment provoked a significant decrease (41%) of AGE-related autofluorescence in the AGE-modified porcine retinas. Furthermore, treatment of human neural retinas resulted in significant decreases of autofluorescence (&minus, 24%). FN3K-treated murine eyes showed less drusenoid material. Pairwise comparison of drusen on tissue sections revealed significant changes in color intensity after FN3K treatment. NIR microspectroscopy uncovered clear spectral differences in drusenoid material (Bruch&rsquo, s membrane) and drusen after FN3K treatment. Ex vivo treatment strongly reduced size of subretinal drusenoid lesions on OCT imaging (up to 83%). In conclusion, our study demonstrated for the first time a potential role of FN3K in the disruption of AGE-related retinal autofluorescence, drusenoid material and drusenoid lesions in patients with AMD.

Preoperative radiotherapy (RT) is a mainstay in the management of rectal cancer, a tumor characterized by desmoplastic stroma containing cancer-associated fibroblasts (CAF). Although CAFs are abundantly present, the effects of RT to CAF and its impact on cancer cells are unknown. We evaluated the damage responses of CAF to RT and investigated changes in colorectal cancer cell growth, transcriptome, metabolome, and kinome in response to paracrine signals emerging from irradiated CAF. RT to CAF induced DNA damage, p53 activation, cell-cycle arrest, and secretion of paracrine mediators, including insulin-like growth factor-1 (IGF1). Subsequently, RT-activated CAFs promoted survival of colorectal cancer cells, as well as a metabolic switch favoring glutamine consumption through IGF1 receptor (IGF1R) activation. RT followed by IGF1R neutralization in orthotopic colorectal cancer models reduced the number of mice with organ metastases. Activation of the downstream IGF1R mediator mTOR was significantly higher in matched (intrapatient) samples and in unmatched (interpatient) samples from rectal cancer patients after neoadjuvant chemoradiotherapy. Taken together, our data support the notion that paracrine IGF1/IGF1R signaling initiated by RT-activated CAF worsens colorectal cancer progression, establishing a preclinical rationale to target this activation loop to further improve clinical responses and patient survival. Significance: These findings reveal that paracrine IGF1/IGF1R signaling promotes colorectal cancer progression, establishing a preclinical rationale to target this activation loop. Cancer Res; 78(3); 659–70. ©2017 AACR.

Fanconi Anaemia (FA) is an autosomal recessive disorder characterised by defects in DNA repair, associated with chromosomal instability and cellular hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC). The FA repair pathway involves complex DNA repair mechanisms crucial for genomic stability. Deficiencies in DNA repair genes give rise to chromosomal radiosensitivity. FA patients have shown increased clinical radiosensitivity by exhibiting adverse normal tissue side-effects. The study aimed to investigate chromosomal radiosensitivity of homozygous and heterozygous carriers of FA mutations using three micronucleus (MN) assays. The G0 and S/G2MN assays are cytogenetic assays to evaluate DNA damage induced by ionising radiation in different phases of the cell cycle. The MMC MN assay detects DNA damage induced by a crosslinking agent in the G0 phase. Patients with a clinical diagnosis of FA and their parents were screened for the complete coding region of 20 FA genes. Blood samples of all FA patients and parents were exposed to ionising radiation of 2 and 4Gy. Chromosomal radiosensitivity was evaluated in the G0 and S/G2 phase. Most of our patients were homozygous for the founder mutation FANCG c.637_643delTACCGCC; p.(Tyr213Lysfs*6) while one patient was compound heterozygous for FANCG c.637_643delTACCGCC and FANCG c.1379G > A, p.(Gly460Asp), a novel missense mutation. Another patient was compound heterozygous for two deleterious FANCA mutations. In FA patients, the G0- and S/G2-MN assays show significantly increased chromosomal radiosensitivity and genomic instability. Moreover, chromosomal damage was significantly elevated in MMC treated FA cells. We also observed an increase in chromosomal radiosensitivity and genomic instability in the parents using 3 assays. The effect was significant using the MMC MN assay. The MMC MN assay is advantageous as it is less labour intense, time effective and has potential as a reliable alternative method for detecting FA patients from parents and controls.

Human C3A cells were exposed to extremely low frequency (50 Hz) magnetic fields (ELF-MF’s) up to 500 µT. They were subjected to the micronucleus assay using a Fluorescence In Situ Hybridization (FISH) technique with an in-house pan-centromere probe. We found no increased frequency in micronucleated cells and no change in the proportion of centromere positive over centromere negative micronuclei compared to the unexposed control cells. These results are in accordance with some, but in contradiction with other previously published investigations underlining that effects of environmental ELF-EMF’s on cellular DNA may be very subtle and that small changes or environmental influences may determine the outcome of a (geno)toxicity study. Interestingly, a low-level (5µT) exposure resulted in less than the background micronucleus frequency., Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics, V. 72 N. 2 (2019)

A focused, single outcome meta-analysis on the protective role of extracts of green tea catechins against prostate cancer. Randomized, placebo-controlled studies enrolling patients with a histologically confirmed diagnosis of high-grade Prostate Intraepithelial Neoplasia or Atypical Small Acinar proliferation but no prostate cancer were included. Meta-analysis for binary data was performed using Mantel-Haenszel statistics, using a random-effects model. Heterogeneity was investigated by calculating the I2. Four studies matched the inclusion criteria for the review. The pooled population was 223 patients; 114 and 109 patients were randomized to catechin and placebo groups, respectively. Nine cases of prstate cancer occurred in the catechin arm (7.9%), and 24 cases were reported in the placebo arm (22%). Pooled analysis resulted in a significant reduction of cancer risk in favor of the catechin arm (risk-ratio = 0.41; 95% CI: 0.19- 0.86; I2 = 0). In conclusion, our data suggest that the intake of concentrated green tea catechin preparations may confer a significant protective effect to carriers of early neoplastic lesions in the prostate. The quality of the evidence is moderate, and additional, largescale studies are warranted to substantiate these preliminary findings.

Purpose Even with an optimal treatment protocol, the median survival of glioblastoma (GB) patients is only 12–15 months. Hence, there is need for novel effective therapies that improve survival outcomes. Recent evidence suggests an important role for connexin (Cx) proteins (especially Cx43) in the microenvironment of malignant glioma. Cx43-mediated gap junctional communication has been observed between tumor cells, between astrocytes and between tumor cells and astrocytes. Therefore, gap junction directed therapy using a pharmacological suppressor or modulator, such as tonabersat, could be a promising target in the treatment of GB. In this preclinical study, we evaluated the possible therapeutic potential of tonabersat in the F98 model. Procedures Female Fischer rats were inoculated with ± 25.000 F98 tumor cells in the right frontal lobe. Eight days post-inoculation contrast-enhanced T1-weighted (CE-T1w) magnetic resonance (MR) images were acquired to confirm tumor growth in the brain. After tumor confirmation, rats were randomized into a Control Group, a Connexin Modulation Group (CM), a Standard Medical Treatment Group (ST), and a Standard Medical Treatment with adjuvant Connexin Modulation Group (STCM). To evaluate therapy response, T2-weighted (T2w) and CE-T1w sequences were acquired at several time points. Tumor volume analysis was performed on CE-T1w images and statistical analysis was performed using a linear mixed model. Results Significant differences in estimated geometric mean tumor volumes were found between the ST Group and the Control Group and also between the STCM Group and the Control Group. In addition, significant differences in estimated geometric mean tumor volumes between the ST Group and the STCM Group were demonstrated. No significant differences in estimated geometric mean tumor volumes were found between the Control Group and the CM Group. Conclusion Our results demonstrate a therapeutic potential of tonabersat for the treatment of GB when used in combination with radiotherapy and temozolomide chemotherapy.

Purpose: Based on clinical and molecular data, breast cancer is a heterogeneous disease. Breast cancers that have no expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) are defined as triple negative breast cancers (TNBCs); luminal cancers have different expressions of ER, PR and/or HER2. TNBCs are frequently linked with advanced disease, poor prognosis and occurrence in young African women, and about 15% of the cases are associated with germline BRCA1/2 mutations. Since radiotherapy is utilized as a principle treatment in the management of TNBC, we aimed to investigate the chromosomal instability and radiosensitivity of lymphocytes in TNBC patients compared to luminal breast cancer patients and healthy controls using the micronucleus (MN) assay. The effect of mutations in breast cancer susceptibility genes on chromosomal radiosensitivity was also evaluated. Methods: Chromosomal radiosensitivity was evaluated in the G0 (83 patients and 90 controls) and S/G2 (34 patients and 17 controls) phase of the cell cycle by exposing blood samples from all patients and controls to 2 and 4 Gy ionizing radiation (IR). Results: In the G0 MN assay, the combined cohort of all breast cancer, TNBC and luminal patients' exhibit significantly elevated spontaneous MN values compared to controls indicating chromosomal instability. Chromosomal radiosensitivity is also significantly elevated in the combined cohort of all breast cancer patients compared to controls. The TNBC patients, however, do not exhibit enhanced chromosomal radiosensitivity. Similarly, in the S/G2 phase, 76% of TNBC patients do not show enhanced chromosomal radiosensitivity compared to the controls. In both the G0 and S/G2 phase, luminal breast cancer patients demonstrate a shift toward chromosomal radiosensitivity compared to TNBC patients and controls. Conclusions: The observations of the MN assay suggest increased chromosomal instability and chromosomal radiosensitivity in South African breast cancer patients. However, in TNBC patients, the irradiated MN values are not elevated. Our results suggest that the healthy lymphocytes in TNBC patients could handle higher doses of IR.

Objective: Intestinal inflammation triggers postoperative ileus (POI), commonly seen after abdominal surgery and characterized by impaired gastrointestinal transit; when prolonged, this leads to increased morbidity. Hydrogen sulfide (H2S) is recognized as an important mediator of many (patho) physiological processes, including inflammation, and is now investigated for anti-inflammatory application. Therefore, the aim of this study was to investigate the effect of the H2S-releasing naproxen derivative ATB-346, developed to reduce gastrointestinal injury by naproxen, and the slow-release H2S donor GYY4137 on intestinal inflammation and delayed gastrointestinal transit in murine POI. Methods: C57BI6J mice were fasted for 6 h,anesthetized and after laparotomy, POI was induced by compressing the small intestine with two cotton applicators for 5 min (intestinal manipulation; IM). GYY4137 (50 mg/kg, intraperitoneally), ATB-346 (16 mg/kg, intragastrically) or naproxen (10 mg/kg, intragastrically) were administered 1 h before IM. At 24 h postoperatively, gastrointestinal transit was assessed via fluorescent imaging, and mucosa-free muscularis segments were prepared for later analysis. Inflammatory parameters and activity of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 were measured. Histological examination of whole tissue sections was done on hematoxylin-eosin stained slides. Results: Pre-treatment with GYY4137 (geometric center; GC: 7.6 +/- 0.5) and ATB-346 (GC: 8.4 +/- 0.3) prevented the delayed transit induced by IM (GC: 3.6 +/- 0.5 vs. 9.0 +/- 0.4 in non-operated controls) while naproxen only partially did (GC: 5.9 +/- 0.5; n = 8 for all groups). GYY4137 and ATB-346 significantly reduced the IM-induced increase in muscular myeloperoxidase (MPO) activity and protein levels of interleukin (IL)-6, IL-1 beta and monocyte chemotactic protein 1; the reduction by naproxen was less pronounced and only reached significance for MPO activity and IL-6 levels. All treatments significantly reduced the increase in COX-2 activity caused by IM, whereas only GYY4137 significantly reduced the increase in iNOS activity. Naproxen treatment caused significant histological damage of intestinal villi. Conclusion: The study shows that naproxen partially prevents POI, probably through its inhibitory effect on COX-2 activity. Both ATB-346 and GYY4137 were more effective, the result with GYY4137 showing that H2S per se can prevent POI.

Purpose: Based on clinical and molecular data, breast cancer is a heterogeneous disease. Breast cancers that have no expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) are defined as triple negative breast cancers (TNBCs); luminal cancers have different expressions of ER, PR and/or HER2. TNBCs are frequently linked with advanced disease, poor prognosis and occurrence in young African women, and about 15% of the cases are associated with germline BRCA1/2 mutations. Since radiotherapy is utilized as a principle treatment in the management of TNBC, we aimed to investigate the chromosomal instability and radiosensitivity of lymphocytes in TNBC patients compared to luminal breast cancer patients and healthy controls using the micronucleus (MN) assay. The effect of mutations in breast cancer susceptibility genes on chromosomal radiosensitivity was also evaluated. Methods: Chromosomal radiosensitivity was evaluated in the G0 (83 patients and 90 controls) and S/G2 (34 patients and 17 controls) phase of the cell cycle by exposing blood samples from all patients and controls to 2 and 4 Gy ionizing radiation (IR). Results: In the G0 MN assay, the combined cohort of all breast cancer, TNBC and luminal patients’ exhibit significantly elevated spontaneous MN values compared to controls indicating chromosomal instability. Chromosomal radiosensitivity is also significantly elevated in the combined cohort of all breast cancer patients compared to controls. The TNBC patients, however, do not exhibit enhanced chromosomal radiosensitivity. Similarly, in the S/G2 phase, 76% of TNBC patients do not show enhanced chromosomal radiosensitivity compared to the controls. In both the G0 and S/G2 phase, luminal breast cancer patients demonstrate a shift toward chromosomal radiosensitivity compared to TNBC patients and controls. Conclusions: The observations of the MN assay suggest increased chromosomal instability and chromosomal radiosensitivity in South African breast cancer patients. However, in TNBC patients, the irradiated MN values are not elevated. Our results suggest that the healthy lymphocytes in TNBC patients could handle higher doses of IR.

To identify the multiparametric magnetic resonance imaging (mpMRI) characteristics of normal, benign and malignant conditions in the prostate. Fifty-six histopathological whole-mount radical prostatectomy specimens from ten randomly selected patients with prostate cancer (PC) were matched with corresponding transverse mpMRI slices. The mpMRI was performed prior to biopsy and consisted of T2-weighted imaging (T2-WI), diffusion-weighted imaging (DWI), dynamic contrast-enhanced imaging (DCE) and magnetic resonance spectroscopic imaging (MRSI). In each prostate specimen, a wide range of histopathological conditions were observed. They showed consistent but overlapping characteristics on mpMRI. Normal glands in the transition zone showed lower signal intensity (SI) on T2-WI, lower ADC values and lower citrate peaks on MRSI as compared to the peripheral zone (PZ) due to sparser glandular elements and more prominent collagenous fibres. In the PZ, normal glands were iso-intense on T2-WI, while high SI areas represented cystic atrophy. Mimickers of well-differentiated PC on mpMRI were inflammation, adenosis, HG-PIN and post-atrophic hyperplasia. Each prostate is a unique mix of normal, benign and/or malignant areas that vary in extent and distribution resulting in very heterogeneous characteristics on mpMRI. Understanding the main concepts of this mpMRI-histopathological correlation may increase the diagnostic confidence in reporting mpMRI. • In each prostate specimen a wide range of histopathological conditions was observed. • Interpretation of mpMRI may be difficult because benign conditions may mimic PC. • High signal intensity areas in the PZ on T2-WI represented cystic atrophy. • The TZ showed sparser glands and more collagenous fibres than the PZ.

As hematopoietic stem and progenitor cells (HSPCs) self-renew throughout life, accumulation of genomic alterations can potentially give rise to radiation carcinogenesis. In this study we examined DNA double-strand break (DSB) induction and repair as well as mutagenic effects of ionizing radiation in CD34(+) cells and T lymphocytes from the umbilical cord of newborns. The age dependence of DNA damage repair end points was investigated by comparing newborn T lymphocytes with adult peripheral blood T lymphocytes. As umbilical cord blood (UCB) contains T lymphocytes that are practically all phenotypically immature, we examined the radiation response of separated naive (CD45RA(+)) and memory (CD45RO(+)) T lymphocytes. The number of DNA DSBs was assessed by microscopic scoring of γ-H2AX/53BP1 foci 0.5 h after low-dose radiation exposure, while DNA repair was studied by scoring the number of residual γ-H2AX/53BP1 foci 24 h after exposure. Mutagenic effects were studied by the cytokinesis block micronucleus (CBMN) assay. No significant differences in the number of DNA DSBs induced by low-dose (100-200 mGy) radiation were observed among the three different cell types. However, residual γ-H2AX/53BP1 foci levels 24 h postirradiation were significantly lower in CD34(+) cells compared to newborn T lymphocytes, while newborn T lymphocytes showed significantly higher foci yields than adult T lymphocytes. No significant differences in the level of radiation-induced micronuclei at 2 Gy were observed between CD34(+) cells and newborn T lymphocytes. However, newborn T lymphocytes showed a significantly higher number of micronuclei compared to adult T lymphocytes. These results confirm that CD34(+) cell quiescence promotes mutagenesis after exposure. Furthermore, we can conclude that newborn peripheral T lymphocytes are significantly more radiosensitive than adult peripheral T lymphocytes. Using the results from the comparative study of radiation-induced DNA damage repair end points in naive (CD45RA(+)) and memory (CD45RO(+)) T lymphocytes, we could demonstrate that the observed differences between newborn and adult T lymphocytes can be explained by the immunophenotypic change of T lymphocytes with age, which is presumably linked with the remodeling of the closed chromatin structure of naive T lymphocytes.

The aim of the present study was to perform cytogenetic analysis by means of a semi-automated micro-nucleus-centromere assay in lymphocytes from medical radiation workers. Two groups of workers receiving the highest occupational doses were selected: 10 nuclear medicine technicians and 10 interventional radiologists/cardiologists. Centromere-negative micronucleus (MNCM−) data, obtained from these two groups of medical radiation workers were compared with those obtained in matched controls. The blood samples of the matched controls were additionally used to construct a 'low-dose' (0–100 mGy) MNCM− dose-response curve to evaluate the sensitivity and suitability of the micronucleus-centromere assay as an 'effect' biomarker in medical surveillance programs. The physical dosimetry data of the 3 years preceding the blood sampling, based on single or double dosimetry practices, were collected for the interpretation of the micronucleus data. The in vitro radiation results showed that for small sized groups, semi-automated scoring of MNCM− enables the detection of a dose of 50 mGy. The comparison of MNCM− yields in medical radiation workers and control individuals showed enhanced MNCM− scores in the medical radiation workers group (P=0.15). The highest MNCM− scores were obtained in the interventional radiologists/cardiologists group, and these scores were significantly higher compared with those obtained from the matched control group (P=0.05). The higher MNCM− scores observed in interventional radiologists/cardiologists compared with nuclear medicine technicians were not in agreement with the personal dosimetry records in both groups, which may point to the limitation of 'double dosimetry' procedures used in interventional radiology/cardiology. In conclusion, the data obtained in the present study supports the importance of cytogenetic analysis, in addition to physical dosimetry, as a routine biomonitoring method in medical radiation workers receiving the highest occupational radiation burdens.

The cytokinesis-block micronucleus (CBMN) assay is a standardized method used for genotoxicity studies. Conventional whole blood cultures (WBC) are often used for this assay, although the assay can also be performed on isolated peripheral blood mononuclear cell (PBMC) cultures. However, the standardization of a protocol for the PBMC CBMN assay has not been investigated extensively. The aim of this study was to optimize a reliable CBMN assay protocol for fresh and cryopreserved peripheral blood mononuclear cells (PBMCS), and to compare micronuclei (MNi) results between WBC and PBMC cultures. The G0 CBMN assay was performed on whole blood, freshly isolated, and cryopreserved PBMCS from healthy human blood samples and five radiosensitive patient samples. Cells were exposed to 220 kV X-ray in vitro doses ranging from 0.5 to 2 Gy. The optimized PBMC CBMN assay showed adequate repeatability and small inter-individual variability. MNi values were significantly higher for WBC than for fresh PBMCS. Additionally, cryopreservation of PBMCS resulted in a significant increase of MNi values, while different cryopreservation times had no significant impact. In conclusion, our standardized CBMN assay on fresh and cryopreserved PBMCS can be used for genotoxicity studies, biological dosimetry, and radiosensitivity assessment.

Purpose Cd is considered as a genotoxic carcinogen for which a threshold can be identified. This threshold has, however, not been established and the shape of the relationship between Cd exposure and genotoxic effects is unknown. The aim of the present study was to analyse the shape of the dose-response relationship for the genotoxic effects of Cd in occupational settings. Methods The study has a cross-sectional design and includes 60 healthy male and female workers with known Cd exposure selected from two plants manufacturing or recycling nickel-Cd batteries. The frequency of MN was measured in circulating lymphocytes, and related to internal Cd doses (Cd-B, Cd-U). Determinants of MN frequency were traced by multivariate regression analysis. Results Cd exposure covered a wide range as measured by Cd-B (0.02–1.26 μg/dL), Cd-U (0.26–15.80 μg/g creat) and seniority in the plant (1–42 years). Gender was the only parameter significantly associated with MN frequency, women having on average 8.5 additional MN/1000 BN cells compared to men. Cd-B, Cd-U or Ni-U did not influence MN frequency when adjusted for gender and other potential confounders. Conclusion This finding is consistent with the existing knowledge on the mechanisms governing the genotoxic activity of Cd, which are all non-stochastic and thresholded. The threshold for systemic genotoxic effects of Cd is thus beyond the range of internal exposure considered in the present investigation.

Diffusion magnetic resonance imaging biomarkers can provide quantifiable information of the brain tissue after a mild traumatic brain injury (mTBI). However, the commonly applied diffusion tensor imaging (DTI) model is not very specific to changes in the underlying cellular structures. To overcome these limitations, other diffusion models have recently emerged to provide a more complete view on the damage profile following TBI. In this study, we investigated longitudinal changes in advanced diffusion metrics following experimental mTBI, utilising three different diffusion models in a rat model of mTBI, including DTI, diffusion kurtosis imaging and a white matter model. Moreover, we investigated the association between the diffusion metrics with histological markers, including glial fibrillary acidic protein (GFAP), neurofilaments and synaptophysin in order to investigate specificity. Our results revealed significant decreases in mean diffusivity in the hippocampus and radial diffusivity and radial extra axonal diffusivity (RadEAD) in the cingulum one week post injury. Furthermore, correlation analysis showed that increased values of fractional anisotropy one day post injury in the hippocampus was highly correlated with GFAP reactivity three months post injury. Additionally, we observed a positive correlation between GFAP on one hand and the kurtosis parameters in the hippocampus on the other hand three months post injury. This result indicated that prolonged glial activation three months post injury is related to higher kurtosis values at later time points. In conclusion, our findings point out to the possible role of kurtosis metrics as well as metrics from the white matter model as prognostic biomarker to monitor prolonged glial reactivity and inflammatory responses after a mTBI not only at early timepoints but also several months after injury., Highlights • Advanced diffusion metrics show longitudinal changes following mTBI • Radial diffusivity (RD) and radial extra-axonal diffusivity ↓ in the cingulum • Mean diffusivity ↓ in the hippocampus • In the cingulum RD is continuously decreased until three months post injury • Glial activity correlates with fractional anisotropy in hippocampus

Breast tissue is very sensitive to ionizing radiation due to the presence of reproductive hormones, including estrogen. In the present pilot study, the efficiency of mammography X-rays to induce DNA double strand breaks (DSB) in mammary epithelial cells was investigated. For this, freshly resected healthy breast tissue was irradiated with 30 kV mammography X-rays in the dose range 0–500 mGy (2, 4, 10, 20, 40, 100 and 500 mGy). Breast specimens were also irradiated with identical doses of 60Co γ-rays as a radiation quality standard. With the γH2AX-foci assay, the number of DNA DSB induced by radiation were quantified in the mammary epithelial cells present in breast tissue. Results indicated that foci induced by 30 kV X-rays and γ-rays followed a biphasic linear dose-response. For 30 kV X-rays, the slope in the low dose region (0–20 mGy) was 8.71 times steeper compared with the slope in the higher dose region (20–500 mGy). Furthermore, compared with γ-rays, 30 kV X-rays were also more effective in inducing γH2AX-foci. This resulted in a relative biological effectiveness (RBE) value of 1.82 in the low dose range. In the higher dose range, an RBE close to 1 was obtained. In conclusion, the results indicated the existence of a low dose hypersensitive response for DSB induction in the dose range representative for mammography screening, which is probably caused by the bystander effect. This could affect the radiation risk calculations for women participating in mammography screening.

For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G > C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies.

When a cancer type proves to be radioresistant to treatment with X-rays, the use of neutrons may constitute therapeutic gain provided the cells are relatively sensitive to high-LET radiation. In this work studies with different tumor cell types are reported following exposure to either photons or different neutron energies used in clinical radiation therapy. Potential for therapeutic gain is clearly noted for neutrons with a mean energy of 6 MeV whilst that for 29 MeV neutrons is dependent on the cell types used in the study.

Background This study characterizes the second hit spectrum in BRCA1 and BRCA2-associated breast and ovarian cancers at both gene loci to investigate if second hit mechanisms are mutually exclusive or able to coincide within the same tumor. Methods Loss of heterozygosity, somatic point mutations and copy number alterations along with promoter methylation were studied in 56 breast and 15 ovarian cancers from BRCA1 and BRCA2 germline mutation carriers. A mathematical methodology was introduced to quantify the tumor cell population carrying a second hit. Results Copy neutral LOH was the most prevalent LOH mechanism in this cohort (BC 69%, OC 67%). However, only 36% of BC and 47% of OC showed LOH in all cancerous cells. Somatic intragenic deletions and methylated subclones were also found in combination with (partial) loss of heterozygosity. Unequivocal deleterious somatic point mutations were not identified in this cohort. Conclusion Different mechanisms inactivating the wild type allele are present within the same tumor sample at various extents. Results indicate that BRCA1/2-linked breast and ovarian cancer cells are predominantly characterized by LOH, but harbor a complex combination of second hits at various frequencies.

International audience; Purpose: A European network was initiated in 2012 by 23 partners from 16 European countries with the aim to significantly increase individualized dose reconstruction in case of large-scale radiological emergency scenarios. Results: The network was built on three complementary pillars: (1) an operational basis with seven biological and physical dosimetric assays in ready-to-use mode, (2) a basis for education, training and quality assurance, and (3) a basis for further network development regarding new techniques and members. Techniques for individual dose estimation based on biological samples and/or inert personalized devices as mobile phones or smart phones were optimized to support rapid categorization of many potential victims according to the received dose to the blood or personal devices. Communication and cross-border collaboration were also standardized. To assure long-term sustainability of the network, cooperation with national and international emergency preparedness organizations was initiated and links to radiation protection and research platforms have been developed. A legal framework, based on a Memorandum of Understanding, was established and signed by 27 organizations by the end of 2015. Conclusions: RENEB is a European Network of biological and physical-retrospective dosimetry, with the capacity and capability to perform large-scale rapid individualized dose estimation. Specialized to handle large numbers of samples, RENEB is able to contribute to radiological emergency preparedness and wider large-scale research projects. © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

International audience; Purpose The European Network of Biological and Physical Retrospective Dosimetry ‘RENEB’ has contributed to European radiation emergency preparedness. To give homogeneous dose estimation results, RENEB partners must harmonize their processes. Materials and methods A first inter-comparison focused on biological and physical dosimetry was used to detect the outliers in terms of dose estimation. Subsequently, trainings were organized to improve both tools dose estimation. A second inter-comparison was performed to validate training efficiency. Simultaneously, based on ISO standards, a QAandQM manual on all dosimetry assays was produced which states a common basis and harmonized procedures for each assay. The evaluation of the agreement of RENEB partners to follow the QAandQM manual was performed through a questionnaire. The integration of new members into the network was carried out in the same way, whatever the assays. Results The training courses on biological and physical dosimetry were judged to be successful because most of the RENEB members’ dose estimates improved in the second inter-comparison. The QAandQM manual describes the consensus for the minimum requirements and the performance criteria for both dosimetry assays. The questionnaire revealed that the whole network capacity currently can manage between 15 and 3800 samples once. Conclusion The methodology used to harmonize all dosimetry practice within the network RENEB was highly successful. The network is operational to manage a mass casualty radiation accident for immediate dose assessment. © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor and Francis Group.

International audience; Purpose: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event. Materials and methods: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners. Results: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes). Conclusions: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested. © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.