Characterization Of Radiation-Induced Lung Fibrosis And Mode Of Cell Death Using Single And Multi-Pulsed Proton Flash Irradiation

Radiation induced toxicity is the primary limiter to dose escalation in radiation therapy. Recent preclinical studies indicate a reduction of normal tissue toxicity using ultra-high dose-rate (FLASH) radiation on the order of 40 Gy/sec or higher, with comparable tumor control. The resulting therapeutic window enhancement has exciting implications for cancer care, but the results thus far are limited to electrons in an energy range with minimal utility for human treatment. Some clinical proton therapy systems are capable of FLASH dose rates at depths which would provide access to most tumor sites. Therefore, demonstrating therapeutic window enhancement for proton FLASH would have significant implications on clinical translation of this novel therapy paradigm. This study presents a comparison of various biological endpoints comparing three modes of irradiation, conventional, FLASH, and so-called pulsed-FLASH. The thorax region of age and sex matched C57/BL6 mice were irradiated to doses of 15, 17.5, 20 Gy on a clinical pencil-beam-scanning proton system using conventional, FLASH, and pulsed-FLASH (0.5 Gy/sec, 40 Gy/sec, 40 Gy/sec at 10% duty cycle respectively), then sacrificed at 8, 16, 24 and 36 week timepoints. Over the course of animal husbandry, the animals were monitored for weight, radiation induced dermatitis, and lung function using barometric plethysmography. A time-to-event analysis quantified the degree of skin damage and breathing function. Upon sacrifice, the harvested lung tissues were either paraffin embedded or frozen. The paraffin embedded samples were stained with H&E and Masson’s Trichrome then evaluated by a board-certified pathologist and by internally developed algorithms to quantify the degree of lung fibrosis. Frozen tissues were sectioned and stained for various modes of cell death and then digitally quantified. Overall, the differences between the treatment types occurred in the higher dose groups, namely 17.5, 20 Gy, with very little difference observed at 15 Gy. Gender differences were also observed for most of the endpoints, namely male mice did not respond differently to dose-rate, whereas female mice in the FLASH group showed better overall survival, lower incidence of dermatitis, better breathing function, and differences in lung pathology. Differences in mode of cell death were also observed between FLASH/pulsed-FLASH and Conventional groups with no apparent gender differences. This study is the first to report the effects of FLASH irradiation using pencil beam proton irradiation. The results of this toxicity study help to corroborate the emerging evidence that ultra-high dose rates help spare healthy tissue from radiation induced toxicity on a platform capable of translation to human trials.