12 Medical Imaging and Radiotherapy Journal (MIRTJ) 39 (1) Medical Imaging and Radiotherapy Journal (MIRTJ) 39 (1) Review article FLASH RADIOTHERAPY AS NEW PERSPECTIVE IN RADIOTHERAPY TECHNOLOGY Flash radioterapija kot nova možnost v radioterapevtski tehnologiji Matej KURALT1, Valerija ŽAGER MARCIUŠ 1,2 1 University of Ljubljana, Faculty of Health Sciences, Department of Medical Imaging and Radiotherapy, Zdravstvena pot 5, 1000 Ljubljana, Slovenia 2 Institute of Oncology Ljubljana, Department of teleradiotherapy, Zaloška ulica 2, 1000 Ljubljana * Corresponding author: vzager@onko-i.si, valerija.zager@zf.uni-lj.si Received: 26. 6. 2022 Accepted: 12. 12. 2022 https://doi.org/10.47724/MIRTJ.2022.i01.a002 ABSTRACT Purpose: The purpose of this article is to present FLASH radiotherapy as a new radiation therapy method, to explain its mechanisms of action, to present possible sources and devices of radiation, and to identify its advantages and disadvantages compared to conventional radiotherapy. Methods: Articles were reviewed for this study in online scientifi c research over the last 10 years (2012–2022). The Preferred Reporting Items for Systematic Reviews and Meta- Analyses fl ow diagram was used to document and report on all decisions made during the study selection process for this review paper. Results and Discussion: Most studies have found that FLASH- RT reduces toxicity to healthy tissue adjacent to a tumour. At present, there is a lack of suitable radiation devices for the use of FLASH-RT, and it will be necessary to adapt existing devices. Conclusion: FLASH-RT could be used in highly radioresistant tumours where CONV-RT would cause too much damage to healthy tissue with an increase in radiation dose. It could also be useful in tumours where CONV-RT is successful but too toxic for healthy tissue adjacent to a tumour. A great deal of research is required before the clinical implementation of FLASH-RT to determine the optimal dose rate, doses for diff erent types of cancer with most the favourable eff ect/ toxicity ratio and technical solution (i.e. radiation source). Keywords: FLASH radiotherapy, radiotherapy, neoplasms, radiotherapy dosage IZVLEČEK Namen: Namen članka je predstaviti FLASH radioterapijo (FLASH-RT) kot novo obsevalno metodo, pojasniti do sedaj znane mehanizme delovanja, predstaviti možne vire in naprave sevanja ter ugotoviti kakšne so njene prednosti in pomanjkljivosti v primerjavi s konvencionalno radioterapijo (CONV-RT). Metode in materiali: Za raziskavo so bili pregledani članki, objavljeni v zadnjih desetih letih (2012-2022) v spletni bazi podatkov. Za sistematični pregled literature in metaanalizo je bil uporabljen diagram za lažji izbor člankov, ki opisujejo značilnosti FLASH-RT. Rezultati in razprava: Pri večini študij je bilo ugotovljeno, da FLASH-RT zmanjša toksičnost na zdrava tkiva ob tumorju. Trenutno je premalo primernih obsevalnih naprav za uporabo FLASH-RT in bo zato potrebno prilagoditi obstoječe naprave. Zaključek: FLASH-RT bi lahko uporabili pri zelo radiorezistentnih tumorjih, kjer bi pri CONV-RT z višjo obsevalno dozo preveč poškodovali zdravo tkivo. Uporabna bi bila tudi pri tumorjih, kjer je CONV-RT uspešna, a ima preveč stranskih učinkov na zdrava tkiva ob tumorju. Pred klinično uporabo bo potrebno napraviti še veliko raziskav in ugotoviti: hitrost doze, dozni odmerek za različne vrste raka in najugodnejše razmerje med učinkom in toksičnostjo ter tehnično rešitev (tj. vir sevanja). Ključne besede: FLASH radioterapija, radioterapija, neoplazme, dozni odmerki v radioterapiji Medical Imaging and Radiotherapy Journal (MIRTJ) 39 (1) 13 INTRODUCTION Radiotherapy is one of the main types of treatment in oncology. In recent decades, a new radiation therapy method called FLASH radiotherapy (FLASH-RT) has been developed, and has been found to have fewer early and late radiation side eff ects, and the same antitumour effi cacy. This is referred to as the FLASH eff ect. This could make FLASH-RT the main radiotherapy method in the future (1, 2). FLASH-RT is defi ned as irradiation with a single ultra-high dose rate (≥ 40 Gy/s) radiotherapy. FLASH irradiation is approximately 400 times faster than conventional irradiation (~5 Gy/min) (1). The FLASH eff ect was fi rst reported by Dewey and Boag in 1959. At that time, they irradiated Serratia marcescens bacteria with 1.5 MV X-rays at ultra-high dose rates. This study showed that bacteria in a nitrogen-oxygen mixture containing 1% oxygen were more radiosensitive than in a 100% nitrogen environment after irradiation at normal dose rates (1000 rad/ min). However, lower radiosensitivity was observed when ultra-high dose rates (10-20 kilorad/2μs) were applied in the same nitrogen-oxygen mixture. Their study thus highlighted the fact that irradiation at ultra-high dose rates can protect bacteria better than conventional radiotherapy (CONV-RT) at normal dose rates (1). FLASH-RT was fi rst used in humans in 2018 at the University Hospital of Lausanne in Switzerland. The patient was a 75-year-old man who was diagnosed with CD30+ T-cell cutaneous lymphoma in 1999. From 2008 to 2018, the patient received CONV-RT, which successfully treated the lymphoma, but experienced severe side eff ects on the skin adjacent to the tumour. In 2018, he was treated with FLASH-RT using a total dose of 15 Gy delivered in 10 x 1 μs pulses (≥ 106 Gy/s, 1.5 Gy per pulse) with a total treatment time of 90 ms. The tumour was initially 3.5 cm in size and started to shrink after 10 days. Complete tumour response was achieved after 36 days and lasted fi ve months. From the beginning, when the irradiated lesion started to shrink, there were only mild redness and minor oedema around the irradiation site, which was diff erent from the patient's problems after conventional irradiation, where the surrounding tissue was more severely damaged and took three to four months to heal (2). Flash-RT mechanism hypotheses There are several diff erent hypotheses regarding the mechanisms of FLASH-RT. However, the exact mechanism of action of FLASH-RT and its eff ects on cells are not yet known. The most commonly used hypotheses to explain the eff ects of FLASH-RT are the oxygen deprivation hypothesis, the role of reactive oxygen species (ROS) and redox reactions, the immune hypothesis and the diff erential response of normal and tumour tissue hypothesis (3). Oxygen defi ciency hypothesis Oxygen is a critical molecule in the biological eff ect of FLASH- RT. It is known that hypoxic tissues are more radioresistant than oxygen-rich tissues. Radiochemical oxygen depletion occurs in FLASH-RT (4). There is an instantaneous consumption of oxygen, which is signifi cantly faster than reoxygenation. Transient radioresistance occurs in healthy tissue due to transient hypoxia. There is thus less toxicity to such tissue (2, 5). This phenomenon is not as pronounced in CONV-RT because the dose rates are lower and repeated several times, so oxygen is replaced in between and the oxygen concentration in the irradiated tissue changes less (4). ROS role hypothesis and redox biology After irradiation with photons and electrons, water is radiolysed and ROS are formed, which cause 60–70% of indirect DNA damage, while 30–40% of the DNA damage is caused by direct interaction between the radiation and the DNA. If there is a lot of oxygen in the tissue, more ROS are produced and more DNA is damaged. This also explains why hypoxic tumours are more radioresistant than well-oxygenated tumours (2). It is also hypothesised that ROS and other free radicals alter biochemical reactions in normal and tumour tissue, and thus contribute to the FLASH eff ect. This was also shown in a study where zebrafi sh embryos were irradiated with FLASH-RT and CONV-RT, and it was determined that there were fewer side eff ects after FLASH-RT. However, when the zebrafi sh were placed in an environment with ROS scavengers one hour before irradiation, no diff erences were identifi ed. They concluded that FLASH-RT increases radioresistance in normal tissue due to a decrease in ROS (1). A study in which zebrafi sh embryos were irradiated with both radiotherapies confi rmed the hypothesis that ROS and other free radicals alter biochemical reactions in tissue (2). Normal and tumour tissue are distinguished both by the generation of free radicals and by the course of redox reactions. The same dose of FLASH-RT as CONV-RT triggers diff erent redox pathways and a lower burden of pro-oxidants because they scavenge free radicals faster than tumour cells. In tumour tissue, peroxidation chain reactions take longer to occur, causing the accumulation of free radicals, resulting in cell damage and destruction (5). Immune hypothesis The FLASH eff ect is thought to be mediated by infl ammatory and immune responses. TGF-beta is important as a pro- infl ammatory cytokine and is thought to be involved in the diff erent eff ect of FLASH-RT compared to CONV-RT. In an in vitro study, the level of TGF-beta in human lung fi broblasts was monitored and found to be less after FLASH-RT with proton beams than with conventional irradiation. The production was only 1.8 times higher in FLASH-RT than in non-irradiated tissue, and 6.5 times higher in CONV-RT, suggesting that FLASH-RT signifi cantly reduced chronic infl ammation relative to CONV-RT (2). Similarly, another study in mice confi rmed that CONV-RT increased the levels of fi ve of the ten cytokines observed, whereas FLASH-RT increased only three. The exact eff ect of TGF-beta is not yet known, but it is thought to be involved in the anti-tumour immune response. It is thought to suppress the immune system and promote cancer progression, increasing the need for inhibitors of the TGF-beta pathway (2). Hypothesis of diff erential response of normal and tumour tissue It was hypothesised that diff erent types of DNA damage after the two irradiations trigger diff erent responses in healthy Kuralt M. et al./ Flash radiotherapy as new perspective in radiotherapy technology 14 Medical Imaging and Radiotherapy Journal (MIRTJ) 39 (1) and tumour tissue. Solid tumours are mostly hypoxic, so they will not be protected from the transient hypoxia induced by FLASH-RT, whereas healthy tissues will be, resulting in a diff erential eff ect. Cancer and normal cells have diff erent abilities to scavenge hydrogen peroxide products (1). It has been found that it is precisely due to diff erent redox metabolism, diff erent levels of ROS and redox metals, such as labile iron, that normal cells scavenge the free radicals generated during irradiation more effi ciently. The authors also found out that cancer cells have higher levels of labile iron and transferrin receptors, which results in an increase in catalytic processes (Fenton reaction) that convert hydrogen peroxide into hydroxyl free radicals, causing more oxidative damage in cancer cells. Healthy cells have less labile iron, and scavenge hydroperoxides formed more rapidly after FLASH-RT (3, 4). Impact on radiotherapy FLASH-RT has the potential to change the theory of radiobiology (1). The fi rst change could be in the fi ve Rs of radiobiology: DNA repair, reoxygenation, repopulation, redistribution and intrinsic radiosensitivity. The duration of FLASH-RT is too short for reoxygenation, repopulation and redistribution to occur, but the eff ect of FLASH-RT may be related to two Rs: DNA repair and intrinsic radiosensitivity (1). Another modifi cation may be the threshold dose to healthy tissue, as pre-clinical studies have confi rmed that a higher dose of FLASH-RT is required to induce the same level of toxicity as CONV-RT. This was confi rmed in a study where CONV-RT irradiation with a dose of 15 Gy induced pulmonary fi brosis, whereas FLASH-RT irradiation with a dose of 20 Gy did not induce the same eff ect, even after 36 weeks. A similar fi nding was made in another study where CONV-RT irradiation at 17 Gy induced severe skin lesions, while FLASH- RT irradiation at 15 and 20 Gy did not. (1). A third option is a comprehensive change in treatment strategy. FLASH-RT can only be performed once for a very short period of time, so concomitant chemoradiotherapy cannot be performed. Only neoadjuvant and adjuvant chemotherapy can be performed (1). The fourth option is a change in the number of fractions in radiotherapy. FLASH-RT is only performed once and could therefore displace CONV-RT (1). Devices and radiation sources In addition to the dose rate and the duration of FLASH-RT, the radiation source is also important. Electrons, photons and protons can be used (1). Most research has used linear accelerator electron beams. These beams are limited to the treatment of superfi cial cancers and intraoperative radiotherapy due to their low penetration and limited energy (4 to 20 MeV) (2). Higher energy electron beams could also be used, i.e. high-energy electron beams with energies of 100 to 250 MeV. Such beams have good depth penetration and are less sensitive to tissue heterogeneity than X-rays (4). Photon beams from linear accelerators are not suffi ciently intense to achieve the required high doses with current technology. However, X-rays from synchrotrons have been successfully used (3). Synchrotron sources have similar beam energies to X-ray tubes, but also have the potential to use spatially fractionated, ultra-high-dose microbeam radiation therapy (MRT). The disadvantage is that synchrotrons are large, expensive and few in number (4). In proton beam radiotherapy, the penetration of the beams is deeper and facilitates the irradiation of deeper tumours. Another advantage is that most of the beam energy is deposited in a narrow area at Bragg's peak, facilitating the precise targeting of the tumour volume while protecting surrounding healthy tissue and organs at risk (2). The aim of this review article is to present FLASH-RT as a new irradiation method, to explain the currently known mechanisms of action, to present possible sources and devices of radiation, and to identify its advantages and disadvantages compared to CONV-RT. METHODS The studies used in this paper were found in online scientifi c research databases and were published in the last 10 years (including 2012 to 2022). To simplify the literature review, we selected some exclusion criteria, such as studies published in the period before 2012, studies that are not in English, papers without full text and papers not related to the theme of our study. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses fl ow diagram was used to document and report on all decisions made during the study selection process for this review paper (Diagram 1). RESULTS AND DISCUSSION The results present a systematic review of irradiation results for studies investigating toxicity to healthy tissue. The essential characteristics expected from FLASH-RT are equal or even higher antitumour effi cacy and lower toxicity to healthy tissue adjacent to a tumour. The eff ects of FLASH-RT have been studied in various animal models of mice, rats, zebrafi sh, pigs and cats, and in organs such as lungs, skin, intestines and brain. The results of in vitro and in vivo studies were also compared. Researchers were also interested in the eff ects of FLASH-RT from diff erent radiation sources. Most reported that there were fewer adverse eff ects on healthy tissue after FLASH-RT compared to CONV-RT (Table 1). In 2014, Favaudon reported that the use of FLASH-RT to treat lung tumours can lead to a complete response, and reduce early and late toxicity aff ecting normal lung tissue. To investigate toxicity, he used healthy mice in which the lungs were irradiated, and the occurrence of pneumonitis and fi brosis was assessed. One group was irradiated with a high single dose of FLASH-RT (≥ 40 Gy/s) and the other group was conventionally irradiated at a dose rate of 0.003 Gy/s. After CONV-RT at 17 Gy, severe pneumonitis and fi brosis occurred in all mice, whereas FLASH-RT at the same dose resulted in neither pneumonitis nor fi brosis, but only at 30 Gy. At 17 Gy, FLASH-RT also prevented TGF-beta activation (6). Similar conclusions were reached by Vozenin et al. (2019), who irradiated the skin of mini-pigs and cats in their study. For FLASH-RT, they used two prototype linear accelerators, the Kinetron (4.5 MeV) and the Oriatron (6 MeV) for the electron source, and a wider range of dose rates. They irradiated 10 equally sized circular patches of skin in each pig. Five diff erent doses ranging from 22 to 34 Gy were used. A dose rate of 5 Gy/min was used for CONV-RT and 300 Gy/s for FLASH-RT. After 36 weeks, skin biopsies were taken. FLASH- Kuralt M. et al./ Flash radiotherapy as new perspective in radiotherapy technology Medical Imaging and Radiotherapy Journal (MIRTJ) 39 (1) 15 Total number of potential scientifi c research papers identifi ed by database search (n=2234) Number of papers identifi ed after duplicate removal (n=1684) Excluded papers (n=728) Published before 2012 Not in English Excluded papers (n=645) Not in full text Not related to the theme Available papers for research (n=1684) Potencial papers for research (n=956) Papers considered suitable for research (n=311) Studies included (n=12) Id en tifi c at io n Sc re en in g In cl ud ed Diagram 1: Selection of documents for systematic review RT had fewer side eff ects: only transient depilation occurred, but hair follicles were preserved. CONV-RT resulted in permanent hair follicle damage, skin fi bronecrosis, epithelial ulceration and hyperkeratosis. In another study, he used cats irradiated for locally advanced squamous cell carcinoma of the nasal planum. A worse antitumour eff ect was observed with CONV-RT. FLASH-RT used a single dose, while diff erent dose rates (from 25 to 41 Gy) were used to fi nd the maximum acceptable dose. They were followed up for 18 months. There was permanent depilation at the irradiation site, but no disturbance of olfaction and nutritional functions. Tumour response was complete after six months and three of the six cats were still disease-free after 18 months. The results of this study are promising because larger mammals were studied and this would be more easily transferable to human research (7). Kuralt M. et al./ Flash radiotherapy as new perspective in radiotherapy technology 16 Medical Imaging and Radiotherapy Journal (MIRTJ) 39 (1) Montay-Gruel et al. (2017) assessed cognitive skills after whole brain irradiation with FLASH-RT and CONV-RT in two separate studies. They used electrons from a linear accelerator for FLASH-RT in the fi rst study, and synchrotron-generated X-ray radiation in the second. They found that FLASH-RT better preserved memory and neurogenesis in the hippocampus, with more than 37% of preserved neurogenesis clusters found in mice after FLASH-RT, but only 14% with CONV-RT. CONV- RT reduced cognitive abilities and signifi cantly reduced cell divisions in the hippocampus (8, 9). Moreover, a study by Alahband (2020) showed that FLASH-RT after the irradiation of mouse brains better preserves the memory, learning and socialisation abilities of these mice for four months after FLASH-RT, whereas CONV-RT impairs these functions. This in turn suggests that FLASH-RT also gives encouraging results in the long term, which would be very good if FLASH-RT were used in the treatment of paediatric patients (10). Diff enderfer (2020) also compared the two proton radiotherapies. He irradiated the abdomen of healthy mice, whole or only part. After FLASH-RT, he found greater cell preservation in intestinal crypts and better crypt regeneration. Analysis of the muscle layer in the intestine also showed less fi brosis after FLASH-RT, or changes comparable to those in non-irradiated mice. The eff ect of proton FLASH-RT on the tumour was then studied. Pancreatic cancer cells were inoculated and this area was irradiated. Both radiotherapies had the same eff ect on the tumour (11). However, a few studies have found that there were more side eff ects after FLASH-RT. Venkatesulu et al. (2019) also observed that both radiotherapies caused lymphopenia, but this was more severe with FLASH-RT. There was even more severe gastrointestinal toxicity after whole abdomen irradiation and the worse survival of mice with FLASH-RT (12). It is diffi cult to compare all studies published to date because the authors do not use the same conditions for both irradiation techniques. Some use electrons as the radiation source for FLASH-RT and photons for CONV-RT. The shape of the irradiation fi eld is also important, as it is diff erent if the irradiation fi eld is circular or square, even if the same area has been irradiated. Vozenin et al. (2019) point out that often in in vitro studies, oxygen concentrations were signifi cantly higher than in vivo. Due to such non-physiological oxygen concentrations (21%), the FLASH eff ect may not occur in these studies, but is observed when concentrations are physiological (3 to 7%) (5). CONCLUSION FLASH-RT is a new irradiation method that was fi rst mentioned in 1959, but has only started to be studied again more intensively in the last two decades. The major benefi ts expected from this method are reduced toxicity to healthy tissue adjacent to a tumour, and an equal or, in some tumour types or conditions, even better antitumour eff ect than in CONV-RT. The mechanism of action of FLASH-RT is not yet fully understood, but there are some hypotheses that try to explain it. Various studies comparing FLASH-RT with CONV-RT are ongoing, but so far only in animals. There is only one known Table 1: Irradiation results for studies investigating toxicity to healthy tissues Author Model Observed variable Total dose (Gy) Dose rate (Gy/s) Modality of radiation Which RT has the advantage?CONV-RT FLASH-RT Favaudon et al. (2014) Mice – Thoracic irradiation Onset of pneumonitis and pulmonary fi brosis 17 ≤ 0.03 ≥ 40 electron FLASH-RT Vozenin et al. (2019) Mini pigs – Skin irradiation Skin toxicity 22-34 0.08 300 electron FLASH-RT Vozenin et al. (2019) Cats – Skin irradiation Skin toxicity 25-41 0.08 300 electron FLASH-RT Montay-Gruel et al. (2017) Mice – Whole brain irradiation Cognitive skills 10 0.1 30– 5.6x106 electron FLASH-RT Montay-Gruel et al. (2018) Mice – Whole brain irradiation Cognitive skills 10 0.05 37 X-ray FLASH-RT Alaghband et al. (2020) Mice (juvenile) – Brain irradiation Cognitive skills 8 7.7×103 4.4×106 electron FLASH-RT Diff enderfer et al. (2020) Mice – Abdomen irradiation Acute cell loss and late fi brosis 12-18 0.5-1 60-100 proton FLASH-RT Venkatesulu et al. (2019) Mice – Heart and spleen irradiation Level of lymphocytes in the circulation 0-8 0.1 35 electron CONV-RT Venkatesulu et al. (2019) Mice – Abdomen irradiation Toxicity 16 0.1 35 electron CONV-RT Kuralt M. et al./ Flash radiotherapy as new perspective in radiotherapy technology Medical Imaging and Radiotherapy Journal (MIRTJ) 39 (1) 17 example of FLASH-RT in humans, which is not suffi cient to translate this method into clinical use. Extensive research is needed before this can be done to optimize the dose rate for diff erent types of cancer, and to determine the dose with the most favourable eff ect/toxicity ratio. It will also be necessary to determine which radiation source is most appropriate for this type of radiation, which will require intensive technological developments in the fi eld of irradiation devices. FLASH-RT could be used for highly radioresistant tumours, where CONV-RT would damage healthy tissue if an increase in radiation dose would be used to overcome radioresistancy. It would also be useful for tumours where CONV-RT is successful in order to further reduce side eff ects on healthy tissue adjacent to a tumour. REFERENCES 1. Lin B, Gao F, Yang Y, Wu D, Zhang Y, Feng G, et al. FLASH Radiotherapy: History and Future. Frontiers in oncology [Internet]. 2021 [cited 2022 Apr 15];11:644400. Available from: https://doi.org/10.3389/fonc.2021.644400 2. Hughes JR, Parsons JL. FLASH Radiotherapy: Current Knowledge and Future Insights Using Proton-Beam Therapy. International journal of molecular sciences [Internet]. 2020 [cited 2022 Apr 15];21(18):6492. 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Venkatesulu BP, Sharma A, Pollard-Larkin JM, Sadagopan R, Symons J, Neri S, et al. Ultra high dose rate (35 Gy/sec) radiation does not spare the normal tissue in cardiac and splenic models of lymphopenia and gastrointestinal syndrome. Scientifi c reports [Internet]. 2019 [cited 2022 Apr 21];9(1), 17180. Available from: https://doi. org/10.1038/s41598-019-53562-y Kuralt M. et al./ Flash radiotherapy as new perspective in radiotherapy technology