Most DNA repair defects do not modify the relationship between relative biological effectiveness and linear energy transfer in CRISPR-edited cells.

BACKGROUND: Cancer is a highly heterogeneous disease, driven by frequent genetic alterations which have significant effects on radiosensitivity. However, radiotherapy for a given cancer type is typically given with a standard dose determined from population-level trials. As a result, a proportion of patients are under- or over-dosed, reducing the clinical benefit of radiotherapy. Biological optimization would not only allow individual dose prescription but also a more efficient allocation of limited resources, such as proton and carbon ion therapy. Proton and ion radiotherapy offer an advantage over photons due to their elevated Relative Biological Effectiveness (RBE) resulting from their elevated Linear Energy Transfer (LET). Despite significant interest in optimizing LET by tailoring radiotherapy plans, RBE's genetic dependence remains unclear. PURPOSE: The aim of this study is to better define the RBE/LET relationship in a panel of cell lines with different defects in DSB repair pathways, but otherwise identical biological features and genetic background to isolate these effects. METHODS: Normal human cells (RPE1), genetically modified to introduce defects in DNA double-strand break (DSB) repair genes, ATM, BRCA1, DCLRE1C, LIG4, PRKDC and TP53, were used to map the RBE-LET relationship. Cell survival was measured with clonogenic assays after exposure to photons, protons (LET 1 and 12 keV/µm) and alpha particles (129 keV/µm). Gene knockout sensitizer enhancement ratio (SER) values were calculated as the ratio of the mean inactivation dose (MID) of wild-type cells to repair-deficient cells, and RBE values were calculated as the ratio of the MID of X-ray and particle irradiated cells. 53BP1 foci were used to quantify radiation-induced DSBs and their repair following irradiation. RESULTS: Deletion of NHEJ genes had the greatest impact on photon sensitivity (ATM-/- SER = 2.0 and Lig4-/- SER = 1.8), with genes associated with HR having smaller effects (BRCA1-/- SER = 1.2). Wild-type cells showed RBEs of 1.1, 1.3, 5.0 for low- and high-LET protons and alpha particles respectively. SERs for different genes were independent of LET, apart from NHEJ knockouts which proved to be markedly hypersensitive across all tested LETs. Due to this hypersensitivity, the impact of high LET was reduced in cell models lacking the NHEJ repair pathway. HR-defective cells had moderately increased sensitivity across all tested LETs, but, notably, the contribution of HR pathway to survival appeared independent of LET. Analysis of 53BP1 foci shows that NHEJ-defective cells had the least DSB repair capacity after low LET exposure, and no visible repair after high LET exposure. HR-defective cells also had slower repair kinetics, but the impact of HR defects is not as severe as NHEJ defects. CONCLUSIONS: DSB repair defects, particularly in NHEJ, conferred significant radiosensitivity across all LETs. This sensitization appeared independent of LET, suggesting that the contribution of different DNA repair pathways to survival does not depend on radiation quality.

High-LET radiation induces large amounts of rapidly-repaired sublethal damage.

There is agreement that high-LET radiation has a high Relative Biological Effectiveness (RBE) when delivered as a single treatment, but how it interacts with radiations of different qualities, such as X-rays, is less clear. We sought to clarify these effects by quantifying and modelling responses to X-ray and alpha particle combinations. Cells were exposed to X-rays, alpha particles, or combinations, with different doses and temporal separations. DNA damage was assessed by 53BP1 immunofluorescence, and radiosensitivity assessed using the clonogenic assay. Mechanistic models were then applied to understand trends in repair and survival. 53BP1 foci yields were significantly reduced in alpha particle exposures compared to X-rays, but these foci were slow to repair. Although alpha particles alone showed no inter-track interactions, substantial interactions were seen between X-rays and alpha particles. Mechanistic modelling suggested that sublethal damage (SLD) repair was independent of radiation quality, but that alpha particles generated substantially more sublethal damage than a similar dose of X-rays, [Formula: see text]. This high RBE may lead to unexpected synergies for combinations of different radiation qualities which must be taken into account in treatment design, and the rapid repair of this damage may impact on mechanistic modelling of radiation responses to high LETs.

Differential responses to 223Ra and Alpha-particles exposure in prostate cancer driven by mitotic catastrophe.

Introduction: Radium-223 (223Ra) has been shown to have an overall survival benefit in metastatic castration-resistant prostate cancer (mCRPC) involving bone. Despite its increased clinical usage, relatively little is known regarding the mechanism of action of 223Ra at the cellular level. Methods: We evaluated the effects of 223Ra irradiation in a panel of cell lines and then compared them with standard X-ray and external alpha-particle irradiation, with a particular focus on cell survival and DNA damage repair kinetics. Results: 223Ra exposures had very high, cell-type-dependent RBE50% ranging from 7 to 15. This was significantly greater than external alpha irradiations (RBE50% from 1.4 to 2.1). These differences were shown to be partially related to the volume of 223Ra solution added, independent of the alpha-particle dose rate, suggesting a radiation-independent mechanism of effect. Both external alpha particles and 223Ra exposure were associated with delayed DNA repair, with similar kinetics. Additionally, the greater treatment efficacy of 223Ra was associated with increased levels of residual DNA damage and cell death by mitotic catastrophe. Conclusions: These results suggest that 223Ra exposure may be associated with greater biological effects than would be expected by direct comparison with a similar dose of external alpha particles, highlighting important challenges for future therapeutic optimization.

DNA Repair Inhibitors Potentiate Fractionated Radiotherapy More Than Single-Dose Radiotherapy in Breast Cancer Cells.

Pharmacological inhibitors of DNA damage response (DDR) proteins, such as the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and Rad3-related (ATR) kinases and poly (ADP-ribose) polymerase (PARP), have been developed to overcome tumor radioresistance. Despite demonstrating radiosensitization preclinically, they have performed suboptimally in clinical trials, possibly due to an incomplete understanding of the influence of DDR inhibition on ionizing radiation (IR) dose fractionation and sublethal damage repair. Hence, this study aimed to evaluate the radiosensitizing ability under fractionation of ATM inhibitor AZD0156, ATR inhibitor AZD6738 and PARP inhibitor AZD2281 (olaparib), utilizing MDA-MB-231 and MCF-7 human breast cancer cells. Clonogenic assays were performed to assess cell survival and sublethal damage repair after treatment with DDR inhibitors and either single-dose or fractionated IR. Immunofluorescence microscopy was utilized to evaluate DNA double-strand break repair kinetics. Cell cycle distributions were investigated using flow cytometry. All inhibitors showed significant radiosensitization, which was significantly greater following fractionated IR than single-dose IR. They also led to more unrepaired DNA double-strand breaks at 24 h post-IR. This study provides preclinical evidence for the role of AZD0156, AZD6738 and olaparib as radiosensitizing agents. Still, it highlights the need to evaluate these drugs in fractionated settings mirroring clinical practice to optimize the trial design.

TOPAS a tool to evaluate the impact of cell geometry and radionuclide on alpha particle therapy.

Due to the increasing clinical application of alpha particles, accurate assessment of their dosimetry at the cellular scale should be strongly advocated. Although observations of the impact of cell and nuclear geometry have been previously reported, this effect has not been fully quantified. Additionally, alpha particle dosimetry presents several challenges and most conventional methodologies have poor resolution and are limited to average parameters across populations of cells. Meaningful dosimetry studies with alpha particles require detailed information on the geometry of the target at a subcellular scale.Methods. The impact of cellular geometry was evaluated for 3 different scenarios, a spherical cell with a concentric nucleus, a spherical cell with an eccentric nucleus and a model of a cell attached to a flask, consisting of a hemispherical oblate ellipsoid, all exposed to 1,700211At radionuclide decays. We also evaluated the cross-irradiation of alpha particles as function of distance to a source cell. Finally, a nanodosimetric analysis of absorbed dose to the nucleus of a cell exposed to 1 Gy of different alpha emitting radionuclides was performed.Results. Simulated data shows the dosimetry of self-absorbed-dose strongly depends on activity localization in the source cell, but that activity localization within the source cell did not significantly affect the cross absorbed dose even when cells are in direct contact with each other. Additionally, nanodosimetric analysis failed to show any significant differences in the energy deposition profile between different alpha particle emitters.Conclusions. The collected data allows a better understanding of the dosimetry of alpha particles emitters at the sub-cellular scale. Dosimetric variations between different cellular configurations can generate complications and confounding factors for the translation of dosimetric outcomes into clinical settings, but effects of different radionuclides are generally similar.

Targeted Alpha Therapy: Current Clinical Applications.

α-Emitting radionuclides have been approved for cancer treatment since 2013, with increasing degrees of success. Despite this clinical utility, little is known regarding the mechanisms of action of α particles in this setting, and accurate assessments of the dosimetry underpinning their effectiveness are lacking. However, targeted alpha therapy (TAT) is gaining more attention as new targets, synthetic chemistry approaches, and α particle emitters are identified, constructed, developed, and realized. From a radiobiological perspective, α particles are more effective at killing cells compared to low linear energy transfer radiation. Also, from these direct effects, it is now evident from preclinical and clinical data that α emitters are capable of both producing effects in nonirradiated bystander cells and stimulating the immune system, extending the biological effects of TAT beyond the range of α particles. The short range of α particles makes them a potent tool to irradiate single-cell lesions or treat solid tumors by minimizing unwanted irradiation of normal tissue surrounding the cancer cells, assuming a high specificity of the radiopharmaceutical and good stability of its chemical bonds. Clinical approval of 223RaCl2 in 2013 was a major milestone in the widespread application of TAT as a safe and effective strategy for cancer treatment. In addition, 225Ac-prostate specific membrane antigen treatment benefit in metastatic castrate-resistant prostate cancer patients, refractory to standard therapies, is another game-changing piece in the short history of TAT clinical application. Clinical applications of TAT are growing with different radionuclides and combination therapies, and in different clinical settings. Despite the remarkable advances in TAT dosimetry and imaging, it has not yet been used to its full potential. Labeled 227Th and 225Ac appear to be promising candidates and could represent the next generation of agents able to extend patient survival in several clinical scenarios.

Mechanistic Modeling of Radium-223 Treatment of Bone Metastases.

PURPOSE: Despite the effectiveness of 223RaCl2 for treating patients with symptomatic bone metastatic disease, its mechanisms of action are still unclear. Even established dosimetric approaches differ considerably in their conclusions. In silico tumor models bring a new perspective to this situation because they can quantitatively simulate the interaction of α-particles with the target(s). Here, we investigated 3 different mathematical models of tumor growth that consider the radiation effect of radium-223 (223Ra) treatments and compared the results with clinical data. METHODS AND MATERIALS: The well-established Gompertz growth model was applied to simulate metastatic tumor burden. On the basis of published measurements of 223Ra uptake, we have incorporated the radiation effect of α-particles into the model and investigated 3 radium distribution scenarios-uniform exposure, exposure of only an outer layer, and exposure of a constant volume of the tumor. For each scenario, the times for various tumor stages to progress to the first symptomatic skeletal event were calculated. RESULTS: Uniform and outer-layer exposure scenarios showed very poor agreement with the Kaplan-Meier patient curves from clinical data. However, the constant-volume effect predicted outcomes very similar to the observed clinical results, suggesting, depending on the dose rate, that relatively small fractions of the cell population see damage from 223Ra. CONCLUSIONS: The commonly used assumption of uniform 223Ra distribution does not accurately reflect clinical responses. The suggestion that only a subpopulation of the tumor might be affected by 223Ra shows a pressing need to further study the tumor and drug kinetics to schedule more effective treatments in the future.

Palliative treatment of metastatic bone pain with radiopharmaceuticals: A perspective beyond Strontium-89 and Samarium-153.

PURPOSE: The present review article aims to provide an overview of the available radionuclides for palliative treatment of bone metastases beyond (89)Sr and (153)Sm. In addition, it aims to review and summarize the clinical outcomes associated with the palliative treatment of bone metastases using different radiopharmaceuticals. MATERIALS AND METHODS: A literature search was conducted on Science Direct and PubMed databases (1990 - 2015). The following search terms were combined in order to obtain relevant results: "bone", "metastases", "palliative", "care", "therapy", "treatment", "radiotherapy", "review", "radiopharmaceutical", "phosphorus-32", "strontium-89", "yttrium-90", "tin-117m", "samarium-153", "holmium-166", "thulium-170", "lutetium-177", "rhenium-186", "rhenium-188" and "radium-223". Studies were included if they provided information regarding the clinical outcomes. RESULTS AND CONCLUSIONS: A comparative analysis of the measured therapeutic response of different radiopharmaceuticals, based on previously published data, suggests that there is a lack of substantial differences in palliative efficacy among radiopharmaceuticals. However, when the comparative analysis adds factors such as patient's life expectancy, radionuclides' physical characteristics (e.g. tissue penetration range and half-life) and health economics to guide the rational selection of a radiopharmaceutical for palliative treatment of bone metastases, (177)Lu and (188)Re-labeled radiopharmaceuticals appear to be the most suitable radiopharmaceuticals for treatment of small and medium/large size bone lesions, respectively.

Comparative analysis of 11 different radioisotopes for palliative treatment of bone metastases by computational methods.

PURPOSE: Throughout the years, the palliative treatment of bone metastases using bone seeking radiotracers has been part of the therapeutic resources used in oncology, but the choice of which bone seeking agent to use is not consensual across sites and limited data are available comparing the characteristics of each radioisotope. Computational simulation is a simple and practical method to study and to compare a variety of radioisotopes for different medical applications, including the palliative treatment of bone metastases. This study aims to evaluate and compare 11 different radioisotopes currently in use or under research for the palliative treatment of bone metastases using computational methods. METHODS: Computational models were used to estimate the percentage of deoxyribonucleic acid (DNA) damage (fast Monte Carlo damage algorithm), the probability of correct DNA repair (Monte Carlo excision repair algorithm), and the radiation-induced cellular effects (virtual cell radiobiology algorithm) post-irradiation with selected particles emitted by phosphorus-32 ((32)P), strontium-89 ((89)Sr), yttrium-90 ((90)Y ), tin-117 ((117m)Sn), samarium-153 ((153)Sm), holmium-166 ((166)Ho), thulium-170 ((170)Tm), lutetium-177 ((177)Lu), rhenium-186 ((186)Re), rhenium-188 ((188)Re), and radium-223 ((223)Ra). RESULTS: (223)Ra alpha particles, (177)Lu beta minus particles, and (170)Tm beta minus particles induced the highest cell death of all investigated particles and radioisotopes. The cell survival fraction measured post-irradiation with beta minus particles emitted by (89)Sr and (153)Sm, two of the most frequently used radionuclides in the palliative treatment of bone metastases in clinical routine practice, was higher than (177)Lu beta minus particles and (223)Ra alpha particles. CONCLUSIONS: (223)Ra and (177)Lu hold the highest potential for palliative treatment of bone metastases of all radioisotopes compared in this study. Data reported here may prompt future in vitro and in vivo experiments comparing different radionuclides for palliative treatment of bone metastases, raise the need for the careful rethinking of the current widespread clinical use of (89)Sr and (153)Sm, and perhaps strengthen the use of (223)Ra and (177)Lu in the palliative treatment of bone metastases.