PurposeTo investigate the clinical implications of a variable relative biological effectiveness (RBE) on proton dose fractionation. Using acute exposures, the current clinical adoption of a generic, constant cell killing RBE has been shown to underestimate the effect of the sharp increase in linear energy transfer (LET) in the distal regions of the spread-out Bragg peak (SOBP). However, experimental data for the impact of dose fractionation in such scenarios are still limited.Methods and MaterialsHuman fibroblasts (AG01522) at 4 key depth positions on a clinical SOBP of maximum energy 219.65 MeV were subjected to various fractionation regimens with an interfraction period of 24 hours at Proton Therapy Center in Prague, Czech Republic. Cell killing RBE variations were measured using standard clonogenic assays and were further validated using Monte Carlo simulations and parameterized using a linear quadratic formalism.ResultsSignificant variations in the cell killing RBE for fractionated exposures along the proton dose profile were observed. RBE increased sharply toward the distal position, corresponding to a reduction in cell sparing effectiveness of fractionated proton exposures at higher LET. The effect was more pronounced at smaller doses per fraction. Experimental survival fractions were adequately predicted using a linear quadratic formalism assuming full repair between fractions. Data were also used to validate a parameterized variable RBE model based on linear α parameter response with LET that showed considerable deviations from clinically predicted isoeffective fractionation regimens.ConclusionsThe RBE-weighted absorbed dose calculated using the clinically adopted generic RBE of 1.1 significantly underestimates the biological effective dose from variable RBE, particularly in fractionation regimens with low doses per fraction. Coupled with an increase in effective range in fractionated exposures, our study provides an RBE dataset that can be used by the modeling community for the optimization of fractionated proton therapy.
Recent ground-breaking developments in Omics have generated new hope for overcoming the complexity and variability of biological systems while simultaneously shedding more light on fundamental radiobiological questions that have remained unanswered for decades. In the era of Omics, our knowledge of how genes and proteins interact in the frame of complex networks to preserve genome integrity has been rapidly expanding. Nevertheless, these functional networks must be observed with strong correspondence to the cell nucleus, which is the main target of ionizing radiation. Nuclear architecture and nuclear processes, including DNA damage responses, are precisely organized in space and time. Information regarding these intricate processes cannot be achieved using high-throughput Omics approaches alone, but requires sophisticated structural probing and imaging. Based on the results obtained from studying the relationship between higher-order chromatin structure, DNA double-strand break induction and repair, and the formation of chromosomal translocations, we show the development of Omics solutions especially for radiation research (radiomics) (discussed in this article) and how confocal microscopy as well as novel approaches of molecular localization nanoscopy fill the gaps to successfully place the Omics data in the context of space and time (discussed in our other article in this issue, "Determining Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: Part B--Structuromics"). Finally, we introduce a novel method of specific chromatin nanotargeting and speculate future perspectives, which may combine nanoprobing and structural nanoscopy to observe structure-function correlations in living cells in real time. Thus, the Omics networks obtained from function analyses may be enriched by real-time visualization of Structuromics.
Recent groundbreaking developments in Omics and bioinformatics have generated new hope for overcoming the complexity and variability of (radio)biological systems while simultaneously shedding more light on fundamental radiobiological questions that have remained unanswered for decades. In the era of Omics, our knowledge of how genes and dozens of proteins interact in the frame of complex signaling and repair pathways (or, rather, networks) to preserve the integrity of the genome has been rapidly expanding. Nevertheless, these functional networks must be observed with strong correspondence to the cell nucleus, which is the main target of ionizing radiation. Information regarding these intricate processes cannot be achieved using high-throughput Omics approaches alone; it requires sophisticated structural probing and imaging. In the first part of this review, the article "Giving Omics Spatiotemporal Dimensions Using Exciting New Nanoscopy Techniques to Assess Complex Cell Responses to DNA Damage: Part A--Radiomics," we showed the development of different Omics solutions and how they are contributing to a better understanding of cellular radiation response. In this Part B we show how high-resolution confocal microscopy as well as novel approaches of molecular localization nanoscopy fill the gaps to successfully place Omics data in the context of space and time. The dynamics of double-strand breaks during repair processes and chromosomal rearrangements at the microscale correlated to aberration induction are explained. For the first time we visualize pan-nuclear nucleosomal rearrangements and clustering at the nanoscale during repair processes. Finally, we introduce a novel method of specific chromatin nanotargeting based on a computer database search of uniquely binding oligonucleotide combinations (COMBO-FISH). With these challenging techniques on hand, we speculate future perspectives that may combine specific COMBO-FISH nanoprobing and structural nanoscopy to observe structure-function correlations in living cells in real-time. Thus, the Omics networks obtained from function analyses may be enriched by real-time visualization of Structuromics.
The research for high-throughput diagnostic tests for victims of radio/nuclear incidents remains ongoing. In this context, we have previously identified candidate genes that predict risk of late-occurring hematologic acute radiation syndrome (HARS) in a baboon model. The goal of the current study was to validate these genes after radiation exposure in humans. We also examined ex vivo relative to in vivo measurements in both species and describe dose-response relationships. Eighteen baboons were irradiated in vivo to simulate different patterns of partial- or total-body irradiation (TBI), corresponding to an equivalent dose of 2.5 or 5 Sv. Human in vivo blood samples were obtained from patients exposed to different dose ranges: diagnostic computerized tomography (CT; 0.004-0.018 Sv); radiotherapy for prostate cancer (0.25-0.3 Sv); and TBI of leukemia patients (2 × 1.5 or 2 × 2 Sv, five patients each). Peripheral whole blood of another five baboons and human samples from five healthy donors were cultivated ex vivo and irradiated with 0-4 Sv. RNA was isolated pairwise before and 24 h after irradiation and converted into cDNA. Gene expression of six promising candidate genes found previously by us in a baboon model ( WNT3, POU2AF1, CCR7, ARG2, CD177, WLS), as well as three genes commonly used in ex vivo whole blood experiments ( FDXR, PCNA, DDB2) was measured using qRT-PCR. We confirmed the six baboon candidate genes in leukemia patients. However, expression for the candidate gene FDXR showed an inverse relationship, as it was downregulated in baboons and upregulated in human samples. Comparisons among the in vivo and ex vivo experiments revealed the same pattern in both species and indicated peripheral blood cells to represent the radiation-responsive targets causing WNT3 and POU2AF1 gene expression changes. CCR7, ARG2, CD177 and WLS appeared to be altered due to radiation-responsive targets other than the whole blood cells. Linear dose-response relationships of FDXR, WNT3 and POU2AF1 using human ex vivo samples corresponded with human in vivo samples, suggesting that ex vivo models for in vivo dose estimates can be used over a wide dose range (0.001-5 Sv for POU2AF1). In summary, we validated six baboon candidate genes in humans, but the FDXR measurements underscored the importance of independent assessments even when candidates from animal models have striking gene sequence homology to humans. Since whole blood cells represented the same radiation-responsive targets for FDXR, WNT3 and POU2AF1 gene expression changes, ex vivo cell culture models can be utilized for in vivo dose estimates over a dose range covering up to 3.5 log scales. These findings might be a step forward in the development of a gene expression-based high-throughput diagnostic test for populations involved in large-scale radio/nuclear incidents.
The increasing risk of acute large-scale radiological/nuclear exposures of population underlines the necessity of developing new, rapid and high throughput biodosimetric tools for estimation of received dose and initial triage. We aimed to compare the induction and persistence of different radiation exposure biomarkers in human peripheral blood in vivo. Blood samples of patients with indicated radiotherapy (RT) undergoing partial body irradiation (PBI) were obtained soon before the first treatment and then after 24 h, 48 h, and 5 weeks; i.e. after 1, 2, and 25 fractionated RT procedures. We collected circulating peripheral blood from ten patients with tumor of endometrium (1.8 Gy per fraction) and eight patients with tumor of head and neck (2.0–2.121 Gy per fraction). Incidence of dicentrics and micronuclei was monitored as well as determination of apoptosis and the transcription level of selected radiation-responsive genes. Since mitochondrial DNA (mtDNA) has been reported to be a potential indicator of radiation damage in vitro, we also assessed mtDNA content and deletions by novel multiplex quantitative PCR. Cytogenetic data confirmed linear dose-dependent increase in dicentrics (p < 0.01) and micronuclei (p < 0.001) in peripheral blood mononuclear cells after PBI. Significant up-regulations of five previously identified transcriptional biomarkers of radiation exposure (PHPT1, CCNG1, CDKN1A, GADD45, and SESN1) were also found (p < 0.01). No statistical change in mtDNA deletion levels was detected; however, our data indicate that the total mtDNA content decreased with increasing number of RT fractions. Interestingly, the number of micronuclei appears to correlate with late radiation toxicity (r2 = 0.9025) in endometrial patients suggesting the possibility of predicting the severity of RT-related toxicity by monitoring this parameter. Overall, these data represent, to our best knowledge, the first study providing a multiparametric comparison of radiation biomarkers in human blood in vivo, which have potential for improving biological dosimetry.
Nowadays, the irradiation methodology in proton therapy is switching from the use of passively scattered beams to active pencil beams due to the possibility of more conformal dose distributions. The dose rates of active pencil beams are much higher than those of passive beams. The purpose of this study was to investigate whether there is any difference in the biological effectiveness of these passive and active irradiation modes. The beam qualities of double scattering and pencil beam scanning were measured dosimetrically and simulated using the Monte Carlo code. Using the medulloblastoma cell line DAOY, we performed an in vitro comparison of the two modes in two positions along the dose–deposition curve plateau and inside the Bragg peak. We followed the clonogenic cell survival, apoptosis, micronuclei, and γH2AX assays as biological endpoints. The Monte Carlo simulations did not reveal any difference between the beam qualities of the two modes. Furthermore, we did not observe any statistically significant difference between the two modes in the in vitro comparison of any of the examined biological endpoints. Our results do not show any biologically relevant differences related to the different dose rates of passive and active proton beams.
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