Purpose: Radiotherapy (RT) remains a cornerstone in breast cancer (BC) treatment. While RT is effective, exposure to surrounding healthy tissues can lead to chromosomal damage and potentially increase the risk of secondary cancers. This study investigated chromosomal damage in circulating lymphocytes of BC patients receiving different RT schemes: ultra-hypofractionation in five fractions (UHF) and moderate hypofractionation in either 10–15 fractions (MHF-low) or 20 fractions (MHF-high). We also aimed to investigate how RT parameters influence MN yields in lymphocytes of BC patients. Additionally, the in vitro chromosomal radiosensitivity of primary and second primary BC patients was evaluated.

Materials and methods: Blood samples were collected pre- and post-RT from 182 primary and 50 second primary BC patients. Chromosomal damage was assessed in lymphocytes using the cytokinesis-block micronucleus (CBMN) assay. To evaluate chromosomal radiosensitivity, all blood samples were irradiated in vitro with 1 Gy of X-ray before performing the CBMN assay. Associations of MN yields with cancer type, RT scheme, lymph node irradiation, boost dose delivery, and irradiated body volume were analyzed using linear regression and linear mixed-effects models.

Results: Our findings indicated no difference in MN yields between primary and second primary BC patients. MHF-high RT scheme was associated with the highest MN induction, while the UHF scheme resulted in the lowest. Additionally, increased chromosomal damage was correlated with higher total tumor doses, larger irradiated volumes, and lymph node irradiation. No significant difference in in vitro chromosomal radiosensitivity was observed between primary and second primary BC patients.

Conclusions: Shorter RT scheme with higher dose per fraction may mitigate cytogenetic damage in circulating lymphocytes, offering potential advantages in long-term safety profiles. In vitro chromosomal radiosensitivity, as assessed by CBMN assay is not a hallmark for second primary BC risk.

Purpose: Due to improved therapy, the life expectancy of cancer patients is increasing, and with this, the incidence of radiotherapy (RT)-induced second primary cancers (SPCs). We investigated whether genetic factors may contribute to SPC susceptibility.

Materials and methods: We collected peripheral blood samples from 232 patients treated by external beam RT for a first primary cancer (FPC) of breast, brain and the lymphatic tissue and from 96 unrelated patients who developed various SPC. Samples from patients with FPC were collected before start of RT and at one or two timepoints after the last RT fraction. Samples from patients with SPC were collected at admission to the hospital and, if the patient was treated by RT, once after the last fraction. Peripheral blood mononuclear cells (PBMCs) were isolated, cryopreserved and shipped to project partners. They were analyzed for RT-induced chromosomal aberrations, micronuclei (MN) and γH2AX foci. In addition, the response of PBMC to an in vitro dose was analyzed at the level of MN and γH2AX foci. Whole genomic DNA of samples collected at admission to the hospital was isolated and sequenced to identify possible single nucleotide polymorphisms that characterize patients with SPC.

Results: The logistics of blood collection, cryopreservation, sample shipments and the demographic and clinical data of the patients are provided. The breast FPC and SPC patients came from Ghent, Belgium and Kielce, Poland. Brain FPC patients came from Kielce and Hodgkin lymphoma FPC patients from Stockholm, Sweden. Differences were seen between patients that came from Ghent and Kielce regarding age at diagnosis, RT dose, chemotherapy treatment and time between FPC and SPC. These differences do not pose problems for reaching the aims of the study.

Conclusions: The data presented here serve as the basis for interpreting the results of the experimental assays that are presented in separate papers.

In previous RENEB interlaboratory comparisons based on the manual scoring of dicentric chromosomes, a tendency for systematic overestimation for doses > 2.5 Gy was found. However, these exercises included only very few doses in the high dose range, and they were heterogeneous in terms of radiation quality and evaluation mode, and comparable only to a limited extent. Here, this presumed deviation was explored by investigating three doses > 2.5 Gy. Blood samples were irradiated (2.56, 3.41 and 4.54 Gy) using a ⁶⁰Co source and sent to 14 member laboratories of the RENEB network, which performed the dicentric chromosome assay (manual and/or semi-automatic scoring) and reported dose estimates. Most participants provided estimates that agreed very well with the physical reference doses and all provided dose estimates were in the correct clinical category (> 2 Gy). The previously observed tendency for a systematic bias across all laboratories was not confirmed. However, tendencies for systematic underestimation were detected for dose estimations for reference doses given in terms of absorbed dose to blood and for some participants, a laboratory-specific trend of systematic under- or overestimation was observed. The importance of regularly performed quality checks for a broad dose range became obvious to avoid misinterpretation of results.

Astronauts travelling in space will be exposed to mixed beams of particle radiation and photons. Exposure limits that correspond to defined cancer risk are calculated by multiplying absorbed doses by a radiation-type specific quality factor that reflects the biological effectiveness of the particle without considering possible interaction with photons. We have shown previously that alpha radiation and X-rays may interact resulting in synergistic DNA damage responses in human peripheral blood lymphocytes but the level of intra-individual variability was high. In order to assess the variability and validate the synergism, blood from two male donors was drawn at 9 time points during 3 seasons of the year and exposed to 0–2 Gy of X-rays, alpha particles or 1:1 mixture of both (half the dose each). DNA damage response was quantified by chromosomal aberrations and by mRNA levels of 3 radiation-responsive genes FDXR, CDKN1A and MDM2 measured 24 h post exposure. The quality of response in terms of differential expression of alternative transcripts was assessed by using two primer pairs per gene. A consistently higher than expected effect of mixed beams was found in both donors for chromosomal aberrations and gene expression with some seasonal variability for the latter. No synergy was detected for alternative transcription.

Reliable dosimetry systems are crucial for radiobiological experiments either to quantify the biological consequences of ionizing radiation or to reproduce results by other laboratories. Also, they are essential for didactic purposes in the field of radiation research. Professional dosemeters are expensive and difficult to use in exposure facilities with closed exposure chambers. Consequently, a simple, inexpensive, battery-driven dosemeter was developed that can be easily built using readily available components. Measurements were performed to validate its readout with photons of different energy and dose rate and to demonstrate the applicability of the dosemeter. It turned out that the accuracy of the dose measurements using the developed dosemeter was better than 10%, which is satisfactory for radiobiological experiments. It is concluded that this dosemeter can be used both for determining the dose rates of an exposure facility and for educational purposes. 

Metaphase spreads stained with Giemsa or painted with chromosome-specific probes by fluorescence in situ hybridisation (FISH) have been in use since long for retrospective dose assessment (biological dosimetry). However, in cases of accidental exposure to ionising radiation, the culturing of lymphocytes to obtain metaphase chromosomes and analysis of chromosomal aberrations is time-consuming and problematic after high radiation doses. Similarly, analysing chromosomal damage in G0/G1 cells or nondividing cells by premature chromosome condensation is laborious. Following large-scale radiological emergencies, the time required for analysis is more important than precision of dose estimate. Painting of whole chromosomes using chromosome-specific probes in interphase nuclei by the FISH technique will eliminate the time required for cell culture and allow a fast dose estimate, provided that a meaningful dose-response can be obtained by scoring the number of chromosomal domains visible in interphase nuclei. In order to test the applicability of interphase FISH for quick biological dosimetry, whole blood from a healthy donor was irradiated with 8 Gy of gamma radiation. Irradiated whole blood was kept for 2 h at 37°C to allow DNA repair and thereafter processed for FISH with probes specific for Chromosomes-1 and 2. Damaged chromosomal fragments, distinguished by extra color domains, were observed in interphase nuclei of lymphocytes irradiated with 8 Gy. These fragments were efficiently detected and quantified by the FISH technique utilising both confocal and single plane fluorescence microscopy. Furthermore, a clear dose-response curve for interphase fragments was achieved following exposure to 0, 1, 2, 4 and 8 Gy of gamma radiation. These results demonstrate interphase FISH as a promising test for biodosimetry and for studying cytogenetic effects of radiation in nondividing cells.

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 or > 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. 

Translocation analysis using fluorescence in situ hybridization (FISH) is the method of choice for dose assessment in case of chronic or past exposures to ionizing radiation. Although it is a widespread technique, unlike dicentrics, the number of FISH-based inter-laboratory comparisons is small. For this reason, although the current Running the European Network of Biological and Physical retrospective Dosimetry (RENEB) inter-laboratory comparison 2021 was designed as a fast response to a real emergency scenario, it was considered a good opportunity to perform an inter-laboratory comparison using the FISH technique to gain further experience. The Bundeswehr Institute of Radiobiology provided peripheral blood samples from one healthy human volunteer. Three test samples were irradiated with blinded doses of 0, 1.2, and 3.5 Gy, respectively. Samples were then sent to the seven participating laboratories. The FISH technique was applied according to the standard procedure of each laboratory. Both, the frequency of translocations and the estimated dose for each sample were sent to the coordinator using a special scoring sheet for FISH. All participants sent their results in due time. However, although it was initially requested to send the results based on the full analysis, evaluating 500 equivalent cells, most laboratories only sent the results based on triage, with a smaller number of analyzed cells. In the triage analysis, there was great heterogeneity in the number of equivalent cells scored. On the contrary, for the full analysis, this number was more homogeneous. For all three samples, one laboratory showed outlier yields compared to the other laboratories. Excluding these results, in the triage analysis, the frequency of translocations in sample no. 1 ranged from 0 to 0.013 translocations per cell, and for samples no. 2 and no. 3 the genomic mean frequency were 0.27 +/- 0.03 and 1.47 +/- 0.14, with a coefficient of variation of 0.29 and 0.23 respectively. Considering only results obtained in the triage analysis for sample no. 1, all laboratories, except one, classified this sample as the non-irradiated one. For sample no. 2, excluding the outlier value, the mean reported dose was 1.74 +/- 0.16 Gy indicating a mean deviation of about 0.5 Gy to the delivered dose of 1.2 Gy. For sample no. 3 the mean dose estimated was 4.21 +/- 0.21 Gy indicating a mean deviation of about 0.7 Gy to the delivered dose of 3.5 Gy. In the frame of RENEB, this is the second FISH-based inter-laboratory comparison. The whole exercise was planned as a response to an emergency, therefore, a triage analysis was requested for all the biomarkers except for FISH. Although a full analysis was initially requested for FISH, most of the laboratories reported only a triage-based result. The main reason is that it was not clearly stated what was required before starting the exercise. Results show that most of the laboratories successfully discriminated unexposed and irradiated samples from each other without any overlap. A good agreement in the observed frequencies of translocations was observed but there was a tendency to overestimate the delivered doses. Efforts to improve the harmonization of this technique and subsequent exercises to elucidate the reason for this trend should be promoted.