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Thu, 01.01.1970
COMET ASSAY MEASURES INDICATE LOWER DNA DAMAGE LEVELS IN WHOLE BLOOD PBLS FOLLOWING EX VIVO ELECTRON FLASH EXPOSURES OVER 0.25-1% OXYGEN.
Abstract
Background and Aims
In vivo studies report the normal tissue sparing effects of FLASH radiotherapy compared to conventional dose rate therapy (CONV). Towards this, it is proposed that FLASH causes a transient/local hypoxia so reducing the level of induced DNA damage for FLASH vs. CONV exposure. Therefore, aim of this study is to assess the levels of DNA damage in whole blood PBLs induced by FLASH vs. CONV irradiation under different oxygen concentrations ex vivo.
Methods
Samples of whole blood were irradiated at different oxygen concentrations (ranging between 0.25 and 21%) with a 6 MeV electron beam at a dose rate of either 2 kGy/s (FLASH) or 0.1 Gy/s (CONV). Induced DNA damage levels in whole blood PBLs were evaluated by alkaline Comet assay after 20 Gy irradiation.
Results
<10% difference was noted between the DNA damage levels induced following FLASH and CONV irradiation under normoxic conditions (21% O2). However, following irradiation over 0.25-1% oxygen, lower levels of DNA damage were clearly induced following FLASH exposure, the difference being significant at 0.5% O2. Furthermore, dose-response studies show the FLASH effect to be lost at ≤10Gy.
Conclusions
This study extends our earlier preliminary findings and substantiates that lower levels of DNA damage are induced following FLASH exposure under low oxygen tension, supporting transient radiation-induced hypoxia/ transient oxygen depletion as a mechanism possibly contributing to the tissue sparing effect of FLASH radiotherapy. Further studies to characterise the induced-damage manifest by FLASH and to dissect/differentiate the exact mechanisms involved (radical-radical recombination vs. transient oxygen depletion) will be presented/discussed.
PRECLINICAL STUDIES WITH PROTON FLASH RADIOTHERAPY: BIOLOGICAL EFFECTS AND POTENTIAL MECHANISMS.
Abstract
Background and Aims
Our group has designed and tested the first system to accurately deliver dosimetrically identical FLASH Proton RT (F-PRT; 60-110 Gy/sec) or Standard Proton RT (S-PRT; 0.5-1 Gy/sec) using double-scattered protons. Our purpose is to identify if F-PRT is superior to S-PRT in protecting normal tissues, while equipotent in controlling tumor growth.
Methods
Masson’s trichrome staining, EdU pulsing and single-cell RNA sequencing (scRNA-seq), were used in this study.
Results
We found that F-PRT preserved a significantly higher percentage of regenerated crypts (p<0.01; EdU pulsing) accompanied by a significant increase in overall survival (p<0.01) compared to the S-PRT following 15Gy of whole-abdomen radiation. Moreover, trichrome staining revealed significantly reduced levels of fibrosis (p<0.001) in the F-PRT treated intestines compared to the high levels observed in the S-PRT group. scRNA-seq on 15Gy F-PRT and S-PRT treated intestines, revealed enrichment of stem/progenitor epithelial cell populations with increased proliferative signatures and expression of genes related to the interferon-alpha signature in epithelial and immune cells post F-PRT treatment compared to the S-PRT. Finally, F-PRT was equipotent with S-PRT in controlling syngeneic pancreatic tumor growth in the same mouse strain.
Conclusions
Our preliminary findings suggest that F-PRT may enhance a regenerative, or facultative stem cell program that is associated with greater and more persistent IFN Type I signaling. Understanding the cellular and molecular basis for the effects of F-PRT provides a framework for clinical application of this novel modality with the potential to improve the therapeutic outcome and quality of life of cancer patients.
IN VIVO QUANTIFICATION OF OXYGEN DEPLETION BY ELECTRON FLASH IRRADIATION
Abstract
Background and Aims
The major hypothesis for the underlying mechanism of normal tissue sparing by FLASH has focused on oxygen depletion, however no experimental data have been presented to support it. The aim of this study was to assess changes in tissue oxygenation in vivo produced by FLASH irradiation.
Methods
Oxygen measurements were performed in vivo and in vitro using the phosphorescence quenching method and molecular probe Oxyphor 2P. The changes in oxygenation were quantified in response to irradiation by a 10 MeV electron beam operating at either ultra-high dose rates (UHDR) reaching 300 Gy/s or at conventional dose rates of 0.1 Gy/s.
Results
In vitro experiments with 5% BSA solutions resulted in oxygen depletion g-values of 0.19-0.21 mmHg/Gy for conventional irradiation and 0.16-0.17 mmHg/Gy for UHDR irradiation. In vivo, the total decrease in oxygen after a single fraction of 20 Gy FLASH irradiation was 2.3±0.3 mmHg in normal tissue and 1.0±0.2 mmHg in tumor tissue (p-value < 0.00001), while no changes in oxygenation were observed from a single fraction of 20 Gy applied at conventional dose rates.
Conclusions
In vitro experiments with 5% BSA solutions resulted in oxygen depletion g-values of 0.19-0.21 mmHg/Gy for conventional irradiation and 0.16-0.17 mmHg/Gy for UHDR irradiation. In vivo, the total decrease in oxygen after a single fraction of 20 Gy FLASH irradiation was 2.3±0.3 mmHg in normal tissue and 1.0±0.2 mmHg in tumor tissue (p-value < 0.00001), while no changes in oxygenation were observed from a single fraction of 20 Gy applied at conventional dose rates.
TEMPORAL RESOLUTION REQUIREMENTS FOR MEASURING THE KINETICS OF OXYGEN DEPLETION DURING FLASH RADIOTHERAPY, BASED ON A 3D COMPUTATIONAL MODEL OF BRAIN VASCULATURE
Abstract
Background and Aims
FLASH has been shown to improve the therapeutic ratio of RT. The mechanism behind this effect has been partially explained by the ROD hypothesis. However, to better understand the contribution of oxygen, it is necessary to measure O2 in-vivo during FLASH irradiation. This study’s goal is to determine the temporal resolution required to accurately measure the rapidly changing oxygen concentration in FLASH RT.
Methods
We conducted a computational simulation of oxygen dynamics using a real vascular model constructed from a public fluorescence microscopy dataset. The dynamic distribution of oxygen tension (po2) was modeled by a PDE considering oxygen diffusion, metabolism, and ROD. The underestimation of ROD due to oxygen recovery was evaluated assuming either complete or partial depletion and a range of parameters such as oxygen diffusion, consumption, vascular po2, and vessel density.
Results
The O2 concentration recovers rapidly after FLASH RT. Assuming a temporal resolution of 0.5s, the estimated ROD is only 50.7% and 36.7% of its actual value in cases of partial and complete depletion. Additionally, the underestimation of ROD strongly depends on the vascular density. To estimate ROD rate with 90% accuracy, temporal resolution on the order of milliseconds is required considering the uncertainty in parameters involved.
Conclusions
The rapid recovery of O2 poses a great challenge for in-vivo ROD measurements in FLASH RT. Temporal resolution on the order of milliseconds is recommended for ROD measurements in the normal tissue. Further work is warranted to investigate whether the same requirements apply to ROD measurements in tumors, given their irregular vasculature.