Enhanced RBE of Particle Radiation Depends on Beam Size in the Micrometer Range.

High-linear energy transfer (LET) radiation, such as heavy ions is associated with a higher relative biological effectiveness (RBE) than low-LET radiation, such as photons. Irradiation with low- and high-LET particles differ in the interaction with the cellular matter and therefore in the spatial dose distribution. When a single high-LET particle interacts with matter, it results in doses of up to thousands of gray (Gy) locally concentrated around the ion trajectory, whereas the mean dose averaged over the target, such as a cell nucleus is only in the range of a Gy. DNA damage therefore accumulates in this small volume. In contrast, up to hundreds of low-LET particle hits are required to achieve the same mean dose, resulting in a quasi-homogeneous damage distribution throughout the cell nucleus. In this study, we investigated the dependence of RBE from different spatial dose depositions using different focused beam spot sizes of proton radiation with respect to the induction of chromosome aberrations and clonogenic cell survival. Human-hamster hybrid (AL) as well as Chinese hamster ovary cells (CHO-K1) were irradiated with focused low LET protons of 20 MeV (LET = 2.6 keV/µm) beam energy with a mean dose of 1.7 Gy in a quadratic matrix pattern with point spacing of 5.4 × 5.4 µm2 and 117 protons per matrix point at the ion microbeam SNAKE using different beam spot sizes between 0.8 µm and 2.8 µm (full width at half maximum). The dose-response curves of X-ray reference radiation were used to determine the RBE after a 1.7 Gy dose of radiation. The RBE for the induction of dicentric chromosomes and cell inactivation was increased after irradiation with the smallest beam spot diameter (0.8 µm for chromosome aberration experiments and 1.0 µm for cell survival experiments) compared to homogeneous proton radiation but was still below the RBE of a corresponding high LET single ion hit. By increasing the spot size to 1.6-1.8 µm, the RBE decreased but was still higher than for homogeneously distributed protons. By further increasing the spot size to 2.7-2.8 µm, the RBE was no longer different from the homogeneous radiation. Our experiments demonstrate that varying spot size of low-LET radiation gradually modifies the RBE. This underlines that a substantial fraction of enhanced RBE originates from inhomogeneous energy concentrations on the µm scale (mean intertrack distances of low-LET particles below 0.1 µm) and quantifies the link between such energy concentration and RBE. The missing fraction of RBE enhancement when comparing with high-LET ions is attributed to the high inner track energy deposition on the nanometer scale. The results are compared with model results of PARTRAC and LEM for chromosomal aberration and cell survival, respectively, which suggest mechanistic interpretations of the observed radiation effects.

RENEB Inter-Laboratory Comparison 2021: The Dicentric Chromosome Assay.

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% (semiautomatic 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.

Inter-laboratory comparison of gene expression biodosimetry for protracted radiation exposures as part of the RENEB and EURADOS WG10 2019 exercise.

Large-scale radiation emergency scenarios involving protracted low dose rate radiation exposure (e.g. a hidden radioactive source in a train) necessitate the development of high throughput methods for providing rapid individual dose estimates. During the RENEB (Running the European Network of Biodosimetry) 2019 exercise, four EDTA-blood samples were exposed to an Iridium-192 source (1.36 TBq, Tech-Ops 880 Sentinal) at varying distances and geometries. This resulted in protracted doses ranging between 0.2 and 2.4 Gy using dose rates of 1.5-40 mGy/min and exposure times of 1 or 2.5 h. Blood samples were exposed in thermo bottles that maintained temperatures between 39 and 27.7 °C. After exposure, EDTA-blood samples were transferred into PAXGene tubes to preserve RNA. RNA was isolated in one laboratory and aliquots of four blinded RNA were sent to another five teams for dose estimation based on gene expression changes. Using an X-ray machine, samples for two calibration curves (first: constant dose rate of 8.3 mGy/min and 0.5-8 h varying exposure times; second: varying dose rates of 0.5-8.3 mGy/min and 4 h exposure time) were generated for distribution. Assays were run in each laboratory according to locally established protocols using either a microarray platform (one team) or quantitative real-time PCR (qRT-PCR, five teams). The qRT-PCR measurements were highly reproducible with coefficient of variation below 15% in ≥ 75% of measurements resulting in reported dose estimates ranging between 0 and 0.5 Gy in all samples and in all laboratories. Up to twofold reductions in RNA copy numbers per degree Celsius relative to 37 °C were observed. However, when irradiating independent samples equivalent to the blinded samples but increasing the combined exposure and incubation time to 4 h at 37 °C, expected gene expression changes corresponding to the absorbed doses were observed. Clearly, time and an optimal temperature of 37 °C must be allowed for the biological response to manifest as gene expression changes prior to running the gene expression assay. In conclusion, dose reconstructions based on gene expression measurements are highly reproducible across different techniques, protocols and laboratories. Even a radiation dose of 0.25 Gy protracted over 4 h (1 mGy/min) can be identified. These results demonstrate the importance of the incubation conditions and time span between radiation exposure and measurements of gene expression changes when using this method in a field exercise or real emergency situation.

MODELING STUDIES ON DICENTRICS INDUCTION AFTER SUB-MICROMETER FOCUSED ION BEAM GRID IRRADIATION.

The biophysical simulation tool PARTRAC contains modules for DNA damage response representing non-homologous end joining of DNA double-strand breaks (DSB) and the formation of chromosomal aberrations. Individual DNA ends from the induced DSB are followed regarding both their enzymatic processing and spatial mobility, as is needed for chromosome aberrations to arise via ligating broken ends from different chromosomes. In particular, by tracking the genomic locations of the ligated fragments and the positions of centromeres, the induction of dicentrics can be modeled. In recent experiments, the impact of spatial clustering of DNA damage on dicentric yields has been assessed in AL human-hamster hybrid cells: Defined numbers of 20 MeV protons (linear energy transfer, LET 2.6 keV/µm), 45 MeV Li ions (60 keV/µm) and 55 MeV C ions (310 keV/µm) focused to sub-µm spot sizes were applied with the ion microbeam SNAKE in diverse grid modes, keeping the absorbed dose constant. The impact of the µm-scaled spatial distribution of DSB (focusing effect) has thus been separated from nm-scaled DSB complexity (LET effect). The data provide a unique benchmark for the model calculations. Model and parameter refinements are described that enabled the simulations to largely reproduce both the LET-dependence and the focusing effect as well as the usual biphasic rejoining kinetics. The predictive power of the refined model has been benchmarked against dicentric yields for photon irradiation.

Sub-micrometer 20MeV protons or 45MeV lithium spot irradiation enhances yields of dicentric chromosomes due to clustering of DNA double-strand breaks.

In conventional experiments on biological effects of radiation types of diverse quality, micrometer-scale double-strand break (DSB) clustering is inherently interlinked with clustering of energy deposition events on nanometer scale relevant for DSB induction. Due to this limitation, the role of the micrometer and nanometer scales in diverse biological endpoints cannot be fully separated. To address this issue, hybrid human-hamster AL cells have been irradiated with 45MeV (60keV/µm) lithium ions or 20MeV (2.6keV/µm) protons quasi-homogeneously distributed or focused to 0.5×1µm(2) spots on regular matrix patterns (point distances up to 10.6×10.6µm), with pre-defined particle numbers per spot to provide the same mean dose of 1.7Gy. The yields of dicentrics and their distribution among cells have been scored. In parallel, track-structure based simulations of DSB induction and chromosome aberration formation with PARTRAC have been performed. The results show that the sub-micrometer beam focusing does not enhance DSB yields, but significantly affects the DSB distribution within the nucleus and increases the chance to form DSB pairs in close proximity, which may lead to increased yields of chromosome aberrations. Indeed, the experiments show that focusing 20 lithium ions or 451 protons per spot on a 10.6µm grid induces two or three times more dicentrics, respectively, than a quasi-homogenous irradiation. The simulations reproduce the data in part, but in part suggest more complex behavior such as saturation or overkill not seen in the experiments. The direct experimental demonstration that sub-micrometer clustering of DSB plays a critical role in the induction of dicentrics improves the knowledge on the mechanisms by which these lethal lesions arise, and indicates how the assumptions of the biophysical model could be improved. It also provides a better understanding of the increased biological effectiveness of high-LET radiation.

Improved normal tissue protection by proton and X-ray microchannels compared to homogeneous field irradiation.

The risk of developing normal tissue injuries often limits the radiation dose that can be applied to the tumour in radiation therapy. Microbeam Radiation Therapy (MRT), a spatially fractionated photon radiotherapy is currently tested at the European Synchrotron Radiation Facility (ESRF) to improve normal tissue protection. MRT utilizes an array of microscopically thin and nearly parallel X-ray beams that are generated by a synchrotron. At the ion microprobe SNAKE in Munich focused proton microbeams ("proton microchannels") are studied to improve normal tissue protection. Here, we comparatively investigate microbeam/microchannel irradiations with sub-millimetre X-ray versus proton beams to minimize the risk of normal tissue damage in a human skin model, in vitro. Skin tissues were irradiated with a mean dose of 2 Gy over the irradiated area either with parallel synchrotron-generated X-ray beams at the ESRF or with 20 MeV protons at SNAKE using four different irradiation modes: homogeneous field, parallel lines and microchannel applications using two different channel sizes. Normal tissue viability as determined in an MTT test was significantly higher after proton or X-ray microchannel irradiation compared to a homogeneous field irradiation. In line with these findings genetic damage, as determined by the measurement of micronuclei in keratinocytes, was significantly reduced after proton or X-ray microchannel compared to a homogeneous field irradiation. Our data show that skin irradiation using either X-ray or proton microchannels maintain a higher cell viability and DNA integrity compared to a homogeneous irradiation, and thus might improve normal tissue protection after radiation therapy.

The effects of ultra-high dose rate proton irradiation on growth delay in the treatment of human tumor xenografts in nude mice.

The new technology of laser-driven ion acceleration (LDA) has shown the potential for driving highly brilliant particle beams. Laser-driven ion acceleration differs from conventional proton sources by its ultra-high dose rate, whose radiobiological impact should be investigated thoroughly before adopting current clinical dose concepts. The growth of human FaDu tumors transplanted onto the hind leg of nude mice was measured sonographically. Tumors were irradiated with 20 Gy of 23 MeV protons at pulsed mode with single pulses of 1 ns duration or continuous mode (∼100 ms) in comparison to controls and to a dose-response curve for 6 MV photons. Tumor growth delay and the relative biological effectiveness (RBE) were calculated for all irradiation modes. The mean target dose reconstructed from Gafchromic films was 17.4 ± 0.8 Gy for the pulsed and 19.7 ± 1.1 Gy for the continuous irradiation mode. The mean tumor growth delay was 34 ± 6 days for pulsed, 35 ± 6 days for continuous protons, and 31 ± 7 days for photons 20 ± 1.2 Gy, resulting in RBEs of 1.22 ± 0.19 for pulsed and 1.10 ± 0.18 for continuous protons, respectively. In summary, protons were found to be significantly more effective in reducing the tumor volume than photons (P < 0.05). Together with the results of previous in vitro experiments, the in vivo data reveal no evidence for a substantially different radiobiology that is associated with the ultra-high dose rate of protons that might be generated from advanced laser technology in the future.

Subdiffusion supports joining of correct ends during repair of DNA double-strand breaks.

The mobility of damaged chromatin regions in the nucleus may affect the probability of mis-repair. In this work, live-cell observation and distance tracking of GFP-tagged DNA damage response protein MDC1 was used to study the random-walk behaviour of chromatin domains containing radiation-induced DNA double-strand breaks (DSB). Our measurements indicate a subdiffusion-type random walk process with similar time dependence for isolated and clustered DSBs that were induced by 20 MeV proton or 43 MeV carbon ion micro-irradiation. As compared to normal diffusion, subdiffusion enhances the probability that both ends of a DSB meet, thus promoting high efficiency DNA repair. It also limits their probability of long-range movements and thus lowers the probability of mis-rejoining and chromosome aberrations.

Low LET protons focused to submicrometer shows enhanced radiobiological effectiveness.

This study shows that enhanced radiobiological effectiveness (RBE) values can be generated focusing low linear energy transfer (LET) radiation and thus changing the microdose distribution. 20 MeV protons (LET = 2.65 keV µm(-1)) are focused to submicrometer diameter at the ion microprobe superconducting nanoprobe for applied nuclear (Kern) physics experiments of the Munich tandem accelerator. The RBE values, as determined by measuring micronuclei (RBE(MN) = 1.48 ± 0.07) and dicentrics (RBE(D) = 1.92 ± 0.15), in human-hamster hybrid (A(L)) cells are significantly higher when 117 protons were focused to a submicrometer irradiation field within a 5.4 × 5.4 µm(2) matrix compared to quasi homogeneous in a 1 × 1 µm(2) matrix applied protons (RBE(MN) = 1.28 ± 0.07; RBE(D) = 1.41 ± 0.14) at the same average dose of 1.7 Gy. The RBE values are normalized to standard 70 kV (dicentrics) or 200 kV (micronuclei) x-ray irradiation. The 117 protons applied per point deposit the same amount of energy like a (12)C ion with 55 MeV total energy (4.48 MeV u(-1)). The enhancements are about half of that obtained for (12)C ions (RBE(MN) = 2.20 ± 0.06 and RBE(D) = 3.21 ± 0.10) and they are attributed to intertrack interactions of the induced damages. The measured RBE values show differences from predictions of the local effect model (LEM III) that is used to calculate RBE values for irradiation plans to treat tumors with high LET particles.