Abstract IA-019: Preclinical studies with proton FLASH radiotherapy in mice and canines: Biological effects, biophysical considerations and potential mechanisms
Authors: Verginadis Ioannis , Velalopoulou Anastasia , Hagan Sarah , Goia Denisa , Shoniyozov Khayrullo , Kim Michele , Diffenderfer Eric , Dong Lei , Metz James , Cengel Keith , Busch Theresa , Minn Andy , Maity Amit , Koumenis Constantinos 
Published: April 2021

Abstract
Multiple studies indicate that ultra-high dose rate, “FLASH” Radiotherapy (F-RT), augments the therapeutic index compared to Standard RT (S-RT) by sparing normal tissue from damage while maintaining tumor responses. We previously reported the first studies using FLASH proton RT (F-PRT), demonstrating that compared to S-PRT, F-PRT spares mice from intestinal epithelium fibrosis and leads to improved overall survival at single doses from 13-16 Gy. Moreover, F-PRT spares a higher percentage of proliferating crypt cells and better maintains the villi architecture 3.5 days post-RT. Importantly, F-PRT was equipotent with S-PRT in controlling syngeneic pancreatic tumors in the same mouse strain. Here, we present new data into potential cellular mechanisms leading to the sparing effect. Using single-cell transcriptomics, we find that F-PRT leads to activation of RIG-I-mediated signaling in the immune cell compartment, which has been linked to normal tissue radioprotection. F-PRT led to higher levels of proliferation genes (Myc, mTOR) in the stem/progenitor cell compartment, consistent with higher proliferative rates. Using a model of epithelial-specific knockout of p53, we find that mice treated with 13.5 Gy S-PRT quickly succumb to acute radiation syndrome while F-PRT significantly improves overall survival. This suggests that F-PRT may preserve a higher proportion of progenitor epithelial cells, leading to reduced fibrosis and long-term toxicity. We also expanded studies to include normal skin damage and control of fibrosarcoma tumors. F-PRT comprehensively decreased toxicity relative to S-PRT, as measured by survival endpoints of mortality/compulsory euthanasia. F-PRT decreased perivascular inflammation, dermal inflammatory infiltrates, and dermal fibrosis on histopathology. Critically, the effect of F-PRT on control of syngeneic sarcomas grown in the leg was indistinguishable from that of S-PRT. Based on these results, we initiated a pilot trial for canine osteosarcoma. To date, we have treated 20 canine patients with a single pre-op fraction of S-PRT or F-PRT. Early results following RNAseq from normal and tumor bone tissue revealed that F-PRT and S-PRT differentially affect myogenesis, adipogenesis, EMT, and inflammatory response. While multiple studies with electron and proton F-RT have demonstrated its normal tissue sparing effects, the mechanism at the molecular level remains elusive. Several models have been proposed, including depletion of oxygen in the vicinity of the DNA, ROS recombination, etc. We will present data from studies of hypoxia and its impact on the FLASH effect and discuss this and alternative models for preferential radioprotection. Our results support the notion that F-PRT significantly augments in the therapeutic window of radiotherapy. Several key questions concerning the mechanism of normal tissue sparing, impact of S-RT vs. F-RT on different tissues and biophysical parameters such as fractionation and optimal dose rates will need to be addressed prior to full clinical implementation of this new modality.
Citation Format: Ioannis Verginadis, Anastasia Velalopoulou, Sarah Hagan, Denisa Goia, Khayrullo Shoniyozov, Michele Kim, Eric Diffenderfer, Lei Dong, James Metz, Keith Cengel, Theresa Busch, Andy Minn, Amit Maity, Constantinos Koumenis. Preclinical studies with proton FLASH radiotherapy in mice and canines: Biological effects, biophysical considerations and potential mechanisms [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-019.