FLASH-RT does not affect chromosome translocations and junction structures beyond that of CONV-RT dose-rates

Paul G Barghouth; Stavros Melemenidis; Pierre Montay-Gruel; Jonathan Ollivier; Vignesh Viswanathan; Patrik G Jorge; Luis A Soto; Brianna C Lau; Cheyenne Sadeghi; Anushka Edlabadkar; Richard Zhang; Ning Ru; Janet E Baulch; Rakesh Manjappa; Jinghui Wang; Marie Le Bouteiller; Murat Surucu; Amy Yu; Karl Bush; Lawrie Skinner; Peter G Maxim; Billy W Loo Jr; Charles L Limoli; Marie-Catherine Vozenin; Richard L Frock

doi:10.1016/j.radonc.2023.109906

FLASH-RT does not affect chromosome translocations and junction structures beyond that of CONV-RT dose-rates

Radiother Oncol. 2023 Nov:188:109906. doi: 10.1016/j.radonc.2023.109906. Epub 2023 Sep 9.

Authors

Paul G Barghouth¹, Stavros Melemenidis¹, Pierre Montay-Gruel², Jonathan Ollivier³, Vignesh Viswanathan¹, Patrik G Jorge⁴, Luis A Soto¹, Brianna C Lau¹, Cheyenne Sadeghi¹, Anushka Edlabadkar¹, Richard Zhang⁵, Ning Ru⁵, Janet E Baulch⁵, Rakesh Manjappa¹, Jinghui Wang¹, Marie Le Bouteiller¹, Murat Surucu¹, Amy Yu¹, Karl Bush¹, Lawrie Skinner¹, Peter G Maxim⁵, Billy W Loo Jr¹, Charles L Limoli⁵, Marie-Catherine Vozenin³, Richard L Frock⁶

Affiliations

¹ Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA.
² Laboratory of Radiation Oncology, Department of Radiation Oncology, Lausanne University Hospital and University of Lausanne, Switzerland; Department of Radiation Oncology, University of California, Irvine, CA 92697-2695, USA.
³ Laboratory of Radiation Oncology, Department of Radiation Oncology, Lausanne University Hospital and University of Lausanne, Switzerland.
⁴ Institute of Radiation Physics/CHUV, Lausanne University Hospital, Switzerland.
⁵ Department of Radiation Oncology, University of California, Irvine, CA 92697-2695, USA.
⁶ Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address: frock@stanford.edu.

PMID: 37690668
PMCID: PMC10591966 (available on 2024-11-01)
DOI: 10.1016/j.radonc.2023.109906

Abstract

Background and purpose: The impact of radiotherapy (RT) at ultra high vs conventional dose rate (FLASH vs CONV) on the generation and repair of DNA double strand breaks (DSBs) is an important question that remains to be investigated. Here, we tested the hypothesis as to whether FLASH-RT generates decreased chromosomal translocations compared to CONV-RT.

Materials and methods: We used two FLASH validated electron beams and high-throughput rejoin and genome-wide translocation sequencing (HTGTS-JoinT-seq), employing S. aureus and S. pyogenes Cas9 "bait" DNA double strand breaks (DSBs) in HEK239T cells, to measure differences in bait-proximal repair and their genome-wide translocations to "prey" DSBs generated after various irradiation doses, dose rates and oxygen tensions (normoxic, 21% O₂; physiological, 4% O₂; hypoxic, 2% and 0.5% O₂). Electron irradiation was delivered using a FLASH capable Varian Trilogy and the eRT6/Oriatron at CONV (0.08-0.13 Gy/s) and FLASH (1x10²-5x10⁶ Gy/s) dose rates. Related experiments using clonogenic survival and γH2AX foci in the 293T and the U87 glioblastoma lines were also performed to discern FLASH-RT vs CONV-RT DSB effects.

Results: Normoxic and physioxic irradiation of HEK293T cells increased translocations at the cost of decreasing bait-proximal repair but were indistinguishable between CONV-RT and FLASH-RT. Although no apparent increase in chromosome translocations was observed with hypoxia-induced apoptosis, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal significant differences in the level of translocations nor in their junction structures. Furthermore, RT dose rate modality on U87 cells did not change γH2AX foci numbers at 1- and 24-hours post-irradiation nor did this affect 293T clonogenic survival.

Conclusion: Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT.

Keywords: Apoptosis; Chromosome translocation; FLASH-RT; HTGTS-JoinT-seq; Hypoxia.

Abstract

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