Comment on 'Modeling for predicting survival fraction of cells after ultra-high dose rate irradiation'.

We comment on the recently published study 'Modeling for predicting survival fraction of cells after ultra-high dose rate irradiation' by Shiraishiet al. While the general approach of the study may be appropriate, we wish to comment on its limitations and point out issues concerning their choice of the benchmarking and fitting data. The approach by the authors could become viable in an extended form once more comprehensive data is available.

Robustness of carbon-ion radiotherapy against DNA damage repair associated radiosensitivity variation based on a biophysical model.

BACKGROUND: Interpatient variation of tumor radiosensitivity is rarely considered during the treatment planning process despite its known significance for the therapeutic outcome. PURPOSE: To apply our mechanistic biophysical model to investigate the biological robustness of carbon ion radiotherapy (CIRT) against DNA damage repair interference (DDRi) associated patient-to-patient variability in radiosensitivity and its potential clinical advantages against conventional radiotherapy approaches. METHODS AND MATERIALS: The "UNIfied and VERSatile bio response Engine" (UNIVERSE) was extended by carbon ions and its predictions were compared to a panel of in vitro and in vivo data including various endpoints and DDRi settings within clinically relevant dose and linear energy transfer (LET) ranges. The implications of UNIVERSE predictions were then assessed in a clinical patient scenario considering DDRi variance. RESULTS: UNIVERSE tests well against the applied benchmarks. While in vitro survival curves were predicted with an R2 > 0.92, deviations from in vivo RBE data were less than 5.6% The conducted paradigmatic patient plan study implies a markedly reduced significance of DDRi based radiosensitivity variability in CIRT (13% change of D 50 ${{D}_{50}}$ in target) compared to conventional radiotherapy (62%) and that boosting the LET within the target further amplifies this robustness of CIRT (8%). In the case of heightened tumor radiosensitivity, a dose de-escalation strategy for photons allows a reduction of the maximum effective dose within the normal tissue (NT) from a D 2 ${{D}_2}$ of 2.65 to 1.64 Gy, which lies below the level found for CIRT ( D 2 ${{D}_2}$  = 2.41 Gy) for the analyzed plan and parameters. However, even after de-escalation, the integral effective dose in the NT is found to be substantially higher for conventional radiotherapy in comparison to CIRT ( D m e a n ${{D}_{mean}}$ of 0.75, 0.46, and 0.24 Gy for the conventional plan, its de-escalation and CIRT, respectively). CONCLUSIONS: The framework offers adequate predictions of in vitro and in vivo radiation effects of CIRT while allowing the consideration of DRRi based solely on parameters derived from photon data. The results of the patient planning study underline the potential of CIRT to minimize important sources of interpatient divergence in therapy outcome, especially when combined with techniques that allow to maximize the LET within the tumor. Despite the potential of de-escalation strategies for conventional radiotherapy to reduce the maximum effective dose in the NT, CIRT appears to remain a more favorable option due to its ability to reduce the integral effective dose within the NT.

Do We Preserve Tumor Control Probability (TCP) in FLASH Radiotherapy? A Model-Based Analysis.

Reports of concurrent sparing of normal tissue and iso-effective treatment of tumors at ultra-high dose-rates (uHDR) have fueled the growing field of FLASH radiotherapy. However, iso-effectiveness in tumors is often deduced from the absence of a significant difference in their growth kinetics. In a model-based analysis, we investigate the meaningfulness of these indications for the clinical treatment outcome. The predictions of a previously benchmarked model of uHDR sparing in the "UNIfied and VERSatile bio response Engine" (UNIVERSE) are combined with existing models of tumor volume kinetics as well as tumor control probability (TCP) and compared to experimental data. The potential TCP of FLASH radiotherapy is investigated by varying the assumed dose-rate, fractionation schemes and oxygen concentration in the target. The developed framework describes the reported tumor growth kinetics appropriately, indicating that sparing effects could be present in the tumor but might be too small to be detected with the number of animals used. The TCP predictions show the possibility of substantial loss of treatment efficacy for FLASH radiotherapy depending on several variables, including the fractionation scheme, oxygen level, and DNA repair kinetics. The possible loss of TCP should be seriously considered when assessing the clinical viability of FLASH treatments.

Biosensor Cell-Fit-HD4D for correlation of single-cell fate and microscale energy deposition in complex ion beams.

We present a protocol for the biosensor Cell-Fit-HD4D. It enables long-term monitoring and correlation of single-cell fate with subcellular-deposited energy of ionizing radiation. Cell fate tracking using widefield time-lapse microscopy is uncoupled in time from confocal ion track imaging. Registration of both image acquisition steps allows precise ion track assignment to cells and correlation with cellular readouts. For complete details on the use and execution of this protocol, please refer to Niklas et al. (2022).

Impact of DNA Repair Kinetics and Dose Rate on RBE Predictions in the UNIVERSE.

Accurate knowledge of the relative biological effectiveness (RBE) and its dependencies is crucial to support modern ion beam therapy and its further development. However, the influence of different dose rates of the reference radiation and ion beam are rarely considered. The ion beam RBE-model within our "UNIfied and VERSatile bio response Engine" (UNIVERSE) is extended by including DNA damage repair kinetics to investigate the impact of dose-rate effects on the predicted RBE. It was found that dose-rate effects increase with dose and biological effects saturate at high dose-rates, which is consistent with data- and model-based studies in the literature. In a comparison with RBE measurements from a high dose in-vivo study, the predictions of the presented modification were found to be improved in comparison to the previous version of UNIVERSE and existing clinical approaches that disregard dose-rate effects. Consequently, DNA repair kinetics and the different dose rates applied by the reference and ion beams might need to be considered in biophysical models to accurately predict the RBE. Additionally, this study marks an important step in the further development of UNIVERSE, extending its capabilities in giving theoretical guidance to support progress in ion beam therapy.

Biosensor for deconvolution of individual cell fate in response to ion beam irradiation.

Clonogenic survival assay constitutes the gold standard method for quantifying radiobiological effects. However, it neglects cellular radiation response variability and heterogeneous energy deposition by ion beams on the microscopic scale. We introduce "Cell-Fit-HD4D" a biosensor that enables a deconvolution of individual cell fate in response to the microscopic energy deposition as visualized by optical microscopy. Cell-Fit-HD4D enables single-cell dosimetry in clinically relevant complex radiation fields by correlating microscopic beam parameters with biological endpoints. Decrypting the ion beam's energy deposition and molecular effects at the single-cell level has the potential to improve our understanding of radiobiological dose concepts as well as radiobiological study approaches in general.

The Impact of Sub-Millisecond Damage Fixation Kinetics on the In Vitro Sparing Effect at Ultra-High Dose Rate in UNIVERSE.

The impact of the exact temporal pulse structure on the potential cell and tissue sparing of ultra-high dose-rate irradiation applied in FLASH studies has gained increasing attention. A previous version of our biophysical mechanistic model (UNIVERSE: UNIfied and VERSatile bio response Engine), based on the oxygen depletion hypothesis, has been extended in this work by considering oxygen-dependent damage fixation dynamics on the sub-milliseconds scale and introducing an explicit implementation of the temporal pulse structure. The model successfully reproduces in vitro experimental data on the fast kinetics of the oxygen effect in irradiated mammalian cells. The implemented changes result in a reduction in the assumed amount of oxygen depletion. Furthermore, its increase towards conventional dose-rates is parameterized based on experimental data from the literature. A recalculation of previous benchmarks shows that the model retains its predictive power, while the assumed amount of depleted oxygen approaches measured values. The updated UNIVERSE could be used to investigate the impact of different combinations of pulse structure parameters (e.g., dose per pulse, pulse frequency, number of pulses, etc.), thereby aiding the optimization of potential clinical application and the development of suitable accelerators.

Combined DNA Damage Repair Interference and Ion Beam Therapy: Development, Benchmark, and Clinical Implications of a Mechanistic Biological Model.

PURPOSE: Our purpose was to develop a mechanistic model that describes and predicts radiation response after combined DNA damage repair interference (DDRi) and particle radiation therapy. METHODS AND MATERIALS: The heterogeneous dose distributions of protons and 4He ions were implemented into the "UNIfied and VERSatile bio-response Engine" (UNIVERSE). Predictions for monoenergetic and mixed fields over clinically relevant dose and linear energy transfer range were compared with experimental in vitro survival data measured in this work as well as data available in the literature, including different cell lines and DDR interferences. Ultimately, UNIVERSE predictions were investigated in a patient plan. RESULTS: UNIVERSE accurately predicts survival of cell lines with and without DDRi in clinical settings of ion beam therapy based only on 3 parameters derived from photon data. With increasing dose or linear energy transfer, the radiosensitizing effect of DDRi decreases, resulting in diminished relative biological effect of ion beam radiation for cells subjected to DDRi in comparison to cells that are not. Similar trends were observed in patient plan recalculations; however, this analysis also suggests that DDRi + particle radiation therapy may better preserve the therapeutic window in comparison to DDRi + photon radiation therapy. CONCLUSIONS: The presented framework represents the first mechanistic model of combined DDRi and particle radiation therapy comprehensively benchmarked in clinically relevant scenarios and a step toward more personalized treatment. It reveals potential differences between DDRi + photon radiation therapy versus DDRi + particle radiation therapy, which have not been described so far. UNIVERSE could aid in appraising the clinical viability of combined administration of radiosensitizing drugs and charged particle therapy, as well as the identification of patients with known DDR deficiencies in the tumor who might benefit from therapy with light ions, freeing limited space at heavy ion therapy centers.

FLASH Dose Rate Helium Ion Beams: First In Vitro Investigations.

PURPOSE: To establish and investigate the effects of dose, linear energy transfer (LET), and O2 concentration on biologic response to ultrahigh dose rate (uHDR; FLASH) helium ion beams compared with standard dose rate (SDR) irradiation. METHODS AND MATERIALS: Beam delivery settings for raster-scanned helium ions at both uHDR and SDR were tuned to achieve >100 Gy/s and ∼0.1 Gy/s, respectively. For both SDR and uHDR, plan optimization and calibration for 10 × 10-mm2 fields were performed to assess in vitro response at an LET range of 4.5 to 16 keV/µm. Clonogenic survival assay was conducted at doses ranging from 2 to 12 Gy in 2 human lung epithelial cell lines (A549 and H1437). Radiation-induced nuclear γH2AX foci (RIF) were assessed in both epithelial cell lines and primary human pulmonary fibroblasts. RESULTS: Average dose rates achieved were 185 Gy/s and 0.12 Gy/s for uHDR and SDR, respectively. No differences in cellular response to SDR versus uHDR were observed for all tested doses at 21% O2, and at 2 and 4 Gy at 1% O2. In contrast, at 1% O2 and a dose threshold of ≳8 Gy cell survival was higher and correlated with reduced nuclear γH2AX RIF signal, indicating FLASH sparing effect in the investigated cell lines irradiated with uHDR compared with SDR. CONCLUSIONS: The first uHDR delivery of raster-scanned particle beams was achieved using helium ions, reaching FLASH-level dose-rates of >100 Gy/s. Baseline oxygen levels and delivered dose (≳8 Gy) play a pivotal role, irrespective of the studied cell lines, for observation of a sparing effect for helium ions.

Deciphering Time-Dependent DNA Damage Complexity, Repair, and Oxygen Tension: A Mechanistic Model for FLASH-Dose-Rate Radiation Therapy.

PURPOSE: Irradiation with ultrahigh dose rates (FLASH) has reemerged as a promising radiation therapy approach to effectively lower potential damage burden on normal tissue without sacrificing tumor control. However, the large number of recent FLASH studies have been conducted under vastly different experimental conditions and circumstances (ie, investigated biological endpoint, radiation quality, and environmental oxygen level), with unverified biological mechanisms of action and unexplored interplay effect of the main dependencies. To facilitate radiobiological investigation of FLASH phenomena and assessment of clinical applicability, we present an extension of the mechanistic radiobiological model "UNified and VERSatile bio response Engine" (UNIVERSE). METHODS AND MATERIALS: The dynamic (time-dependent) extension of UNIVERSE was developed incorporating fundamental temporal mechanisms necessary for dose-rate effect prediction, ie, DNA damage repair kinetics [DDRK], oxygen depletion and reoxygenation during irradiation. Model performance in various experimental conditions is validated based on a large panel of in vitro and in vivo data from the literature. The effect of dose, dose rate, oxygen tension, tissue-type, beam quality and DDRK is analyzed. RESULTS: UNIVERSE adequately reproduces dose-, dose-rate- and oxygen tension-dependent influence on cell killing. For the studied systems, results indicate that the extent of cell/tissue sparing effect, if present at all, strongly depends on DDRK and beam quality used for reference conventional irradiation. A validated mechanistic framework for predicting clinically relevant endpoints comparing conventional and FLASH high-dose-rate effect has been successfully established, relying on time-dependent processing of radiation-induced damage classes taking variable oxygen tension into account. CONCLUSIONS: Highlighted by UNIVERSE itself, the multidimensional nature of this relative sparing effect using high-dose-rate radiation compared with conventional means underlines the importance of robust quantification of biophysical characteristics and consistent, well-documented experimental conditions both in vitro and in vivo before clinical translation. To further elucidate underlying mechanisms and appraise clinical viability, UNIVERSE can provide reliable prediction for biophysical investigations of radiation therapy using ultrahigh dose rate.

Carbon ion dosimetry on a fluorescent nuclear track detector using widefield microscopy.

Fluorescent nuclear track detectors (FNTDs) are solid-state dosimeters used in a wide range of dosimetric and biomedical applications in research worldwide. FNTDs are a core but currently underutilized dosimetry tool in the field of radiation biology which are inherently capable of visualizing the tracks of ions used in hadron therapy. The ions that traverse the FNTD deposit their energy according to their linear energy transfer and transform colour centres to form trackspots around their trajectory. These trackspots have fluorescent properties which can be visualized by fluorescence microscopy enabling a well-defined dosimetric readout with a spatial component indicating the trajectory of individual ions. The current method used to analyse the FNTDs is laser scanning confocal microscopy (LSM). LSM enables a precise localization of track spots in x, y and z however due to the scanning of the laser spot across the sample, requires a long time for large samples. This body of work conclusively shows for the first time that the readout of the trackspots present after 0.5 Gy carbon ion irradiation in the FNTD can be captured with a widefield microscope (WF). The WF readout of the FNTD is a factor ∼10 faster, for an area 2.97 times the size making the method nearly a factor 19 faster in track acquisition than LSM. The dramatic decrease in image acquisition time in WF presents an alternative to LSM in FNTD workflows which are limited by time, such as biomedical sensors which combine FNTDs with live cell imaging.

Modeling Direct and Indirect Action on Cell Survival After Photon Irradiation under Normoxia and Hypoxia.

The demand for personalized medicine in radiotherapy has been met by a surge of mechanistic models offering predictions of the biological effect of ionizing radiation under consideration of a growing number of parameters. We present an extension of our existing model of cell survival after photon irradiation to explicitly differentiate between the damage inflicted by the direct and indirect (radicals-mediated) action of ionizing radiation. Within our approach, we assume that the oxygenation status affects the indirect action. The effect of different concentrations of dimethyl sulfoxide (DMSO), an effective radical scavenger, has been simulated at different dose levels in normoxic and hypoxic conditions for various cell lines. Our model is found to accurately predict experimental data available in literature, validating the assumptions made in our approach. The presented extension adds further flexibility to our model and could act as basis for further developments of our model.

Modeling the Effect of Hypoxia and DNA Repair Inhibition on Cell Survival After Photon Irradiation.

Mechanistic approaches to modeling the effects of ionizing radiation on cells are on the rise, promising a better understanding of predictions and higher flexibility concerning conditions to be accounted for. In this work we modified and extended a previously published mechanistic model of cell survival after photon irradiation under hypoxia to account for radiosensitization caused by deficiency or inhibition of DNA damage repair enzymes. The model is shown to be capable of describing the survival data of cells with DNA damage repair deficiency, both under norm- and hypoxia. We find that our parameterization of radiosensitization is invariant under change of oxygen status, indicating that the relevant parameters for both mechanisms can be obtained independently and introduced freely to the model to predict their combined effect.