Use of the γ-H2AX Assay to Investigate DNA Repair Dynamics Following Multiple Radiation Exposures

Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.

Estimation of fetal and effective dose for CT examinations

Doses from CT examinations are difficult to estimate. However, they are requested more frequently due to the increase in CT examinations. In particular, fetal dose estimations are frequently required for patients who have discovered, subsequent to the examination, that they were pregnant when the examination was conducted. A computer model has been developed to facilitate such dose calculations. This model combines empirical beam data with anatomical information. The model has been verified using thermoluminescent dosemeter (TLD) readings of internal and surface dose from both phantoms and patients, including intrauterine doses for patients undergoing afterloading gynaecological intracavitary treatment. Although only limited experimental data were available, the results indicate that the model accurately predicts uterine doses within acceptable errors. This approach has been validated for fetal dose estimation. The model was also used in a comparison with the nationally available CT dose data from the National Radiological Protection Board (NRPB). The two models were found to be in agreement for fetal dose estimations.

Combined Analysis of Gamma-H2AX/53BP1 Foci and Caspase Activation in Lymphocyte Subsets Detects Recent and More Remote Radiation Exposures

Analysis of gamma-H2AX foci in blood lymphocytes is a promising approach for rapid dose estimation to support patient triage after a radiation accident but has one major drawback: the rapid decline of foci levels post-exposure cause major uncertainties in situations where the exact timing between exposure and blood sampling is unknown. To address this issue, radiation-induced apoptosis (RIA) in lymphocytes was investigated using fluorogenic inhibitors of caspases (FLICA) as an independent biomarker for radiation exposure, which may complement the gamma-H2AX assay. Ex vivo X-irradiated peripheral blood lymphocytes from 17 volunteers showed dose-and time-dependent increases in radiation-induced apoptosis over the first 3 days after exposure, albeit with considerable interindividual variation. Comparison with gamma-H2AX and 53BP1 foci counts suggested an inverse correlation between numbers of residual foci and radiation-induced apoptosis in lymphocytes at 24 h postirradiation (P = 0.007). In T-helper (CD4), T-cytotoxic (CD8) and B-cells (CD19), some significant differences in radiation induced DSBs or apoptosis were observed, however no correlation between foci and apoptosis in lymphocyte subsets was observed at 24 h postirradiation. While gamma-H2AX and 53BP1 foci were rapidly induced and then repaired after exposure, radiation-induced apoptosis did not become apparent until 24 h after exposure. Data from six volunteers with different ex vivo doses and post-exposure times were used to test the capability of the combined assay. Results show that simultaneous analysis of gamma-H2AX and radiation-induced apoptosis may provide a rapid and more accurate triage tool in situations where the delay between exposure and blood sampling is unknown compared to gamma-H2AX alone. This combined approach may improve the accuracy of dose estimations in cases where blood sampling is performed days after the radiation exposure. 

Increased susceptibility to delayed genetic effects of low dose X-irradiation in DNA repair deficient cells

Purpose: To examine whether the levels of micronuclei induction, as a marker for genomic instability in the progeny of X-irradiated cells, correlates with DNA repair function.

Materials and methods: Two repair deficient cell lines (X-ray repair cross-complementing 1 [XRCC1] deficient cell line [EM9] and X-ray repair cross complementing 5 [XRCC5; Ku80] deficient X-ray sensitive Chinese hamster ovary [CHO] cell line [xrs5]) were used in addition to wild-type CHO cells. These cells were irradiated with low doses of X-rays (up to 1 Gy). Seven days after irradiation, micronuclei formed in binucleated cells were counted. To assess the contribution of the bystander effect micronuclei induction was measured in progeny of non-irradiated cells co-cultured with cells that had been irradiated with 1Gy.

Results: The delayed induction of micronuclei in 1 Gy-irradiated cells was observed in normal CHO and EM9 but not in xrs5. In the clone analysis, progenies of xrs5 under bystander conditions showed significantly higher levels of micronuclei, while CHO and EM9 did not.

Conclusion: Genomic instability induced by X-irradiation is associated with DSB (double-strand break) repair, even at low doses. It is also suggested that bystander signals, which lead to genomic instability, may be enhanced when DSB repair is compromised.

Impact of geometric variations on delivered dose in highly focused single vocal cord IMRT

Purpose. To investigate the robustness of single vocal cord intensity modulated radiation therapy (IMRT) treatment plans for set-up errors, respiration, and deformation. Material and methods. Four-dimensional computed tomography (4D-CT) scans of 10 early glottic carcinoma patients, previously treated with conventional techniques, were used in this simulation study. For each patient a pre-treatment 4D-CT was used for IMRT planning, generating a reference dose distribution. Prescribed PTV dose was 66 Gy. The impact of systematic set-up errors was simulated by applying shifts of ± 2 mm to the planning CT scans, followed by dose re-calculation with original beam segments, MUs, etc. Effects of respiration and deformation were determined utilizing extreme inhale and exhale CT scans, and repeat scans acquired after 22 Gy, 44 Gy, and 66 Gy, respectively. All doses were calculated using Monte Carlo dose simulations. Results. Considering all investigated geometrical perturbations, reductions in the clinical target volume (CTV) V95%, D98%, D2%, and generalized equivalent uniform dose (gEUD) were limited to 1.2 ± 2.2%, 2.4 ± 2.9%, 0.2 ± 1.8%, and 0.6 ± 1.1 Gy, respectively. The near minimum dose, D98%, was always higher than 89%, and gEUD always remained higher than 66 Gy. Planned contra-lateral (CL) vocal cord DMean, gEUD, and V40 Gy were 38.2 ± 6.0 Gy, 43.4 ± 5.6 Gy, and 42.7 ± 14.9%. With perturbations these values changed by -0.1 ± 4.3 Gy, 0.1 ± 4.0 Gy, and -1.0 ± 9.6%, respectively. Conclusions. On average, CTV dose reductions due to geometrical perturbations were very low, and sparing of the CL vocal cord was maintained. In a few observations (6 of 103 simulated situations), the near-minimum CTV-dose was around 90%, requiring attention in deciding on a future clinical protocol. 

Accelerated partial breast irradiation (APBI): Are breath-hold and volumetric radiation therapy techniques useful?

Background: In a selective group of patients accelerated partial breast irradiation (APBI) might be applied after conservative breast surgery to reduce the amount of irradiated healthy tissue. The role of volumetric modulated arc therapy (VMAT) and voluntary moderately deep inspiration breath-hold (vmDIBH) techniques in further reducing irradiated healthy – especially heart – tissue is investigated.

Material and methods: For 37 partial breast planning target volumes (PTVs), three-dimensional conformal radiotherapy (3D-CRT) (3 – 5 coplanar or non-coplanar 6 and/or 10 MV beams) and VMAT (two partial 6 MV arcs) plans were made on CTs acquired in free-breathing (FB) and/or in vmDIBH. Dose-volume parameters for the PTV, heart, lungs, and breasts were compared. 

Results: Better dose conformity was achieved with VMAT compared to 3D-CRT (conformity index 1.24 0.09 vs. 1.49 0.20). Non-PTV ipsilateral breast receiving 50% of the prescribed dose was on average reduced by 28% in VMAT plans compared to 3D-CRT plans. Mean heart dose (MHD) reduced from 2.0 (0.1 – 5.1) Gy in 3D-CRT(FB) to 0.6 (0.1 – 1.6) Gy in VMAT(vmDIBH). VMAT is benefi cial for MHD reduction if MHD with 3D-CRT exceeds 0.5Gy. Cardiac dose reduction as a result of VMAT increases with increasing initial MHD, and adding vmDIBH reduces the cardiac dose further. Mean dose to the ipsilateral lung decreased from 3.7 (0.7 – 8.7) to 1.8 (0.5 – 4.0) Gy with VMAT(vmDIBH) compared to 3D-CRT(FB). VMAT resulted in a slight increase in the contralateral breast dose (DMean ) always remaining 1.9 Gy). 

Conclusions: For APBI patients, VMAT improves PTV dose conformity and delivers lower doses to the ipsilateral breast and lung compared to 3D-CRT. This goes at the cost of a slight but acceptable increase of the contralateral breast dose. VMAT reduces cardiac dose if MHD exceeds 0.5 Gy for 3D-CRT. Adding vmDIBH results in a further reduction of heart and ipsilateral lung dose. 

Near-monochromatic high-harmonic radiation from relativistic laser-plasma interactions with blazed grating surfaces

Intense, femtosecond laser interactions with blazed grating targets are studied through experiment and particle-in-cell (PIC) simulations. The high harmonic spectrum produced by the laser is angularly dispersed by the grating leading to near-monochromatic spectra emitted at different angles, each dominated by a single harmonic and its integer-multiples. The spectrum emitted in the direction of the third-harmonic diffraction order is measured to contain distinct peaks at the 9th and 12th harmonics which agree well with two-dimensional PIC simulations using the same grating geometry. This confirms that surface smoothing effects do not dominate the far-field distributions for surface features with sizes on the order of the grating grooves whilst also showing this to be a viable method of producing near-monochromatic, short-pulsed extreme-ultraviolet radiation.

A novel experimental approach to investigate radiolysis processes in liquid samples using collimated radiation sources

Here is detailed a novel and low-cost experimental method for high-throughput automated fluid sample irradiation. The sample is delivered via syringe pump to a nozzle, where it is expressed in the form of a hanging droplet into the path of a beam of ionising radiation. The dose delivery is controlled by an upstream lead shutter, which allows the beam to reach the droplet for a user defined period of time. The droplet is then further expressed after irradiation until it falls into one well of a standard microplate. The entire system is automated and can be operated remotely using software designed in-house, allowing for use in environments deemed unsafe for the user (synchrotron beamlines, for example). Depending on the number of wells in the microplate, several droplets can be irradiated before any human interaction is necessary, and the user may choose up to 10 samples per microplate using an array of identical syringe pumps, the design of which is described here. The nozzles consistently produce droplets of 25.1 ± 0.5 μl.

Radiation to control transgene expression in tumors

Significant evidence has accumulated indicating that certain genes are induced by ionising radiation. An implication of this observation is that their promoter regions include radiation-responsive sequences. These sequences have been isolated in the promoter of several genes including Erg-1, p21/WAF-1, GADD45alpha and t-PA. The mechanism by which radiation induces gene expression remains unclear but involves putative binding sites for selected transcription factors and/or p53. Consensus CC(A/T)6GG sequences have been localized in the Erg-1 promoter and are referred to as serum response elements or CArG elements. The tandem combination of CArG elements has been shown to improve gene expression levels, with a 9-copy motif conferring maximum inducibility. The response of these genes to ionising radiation appears to follow a sigmoid relationship with time and dose. Therapeutic induction of suicide genes and significant cytotoxicity can be achieved at clinically relevant x-rays doses both in vitro and in vivo but was found to be cell-type dependent. Radiation-inducible gene therapy can be potentially enhanced by exploiting hypoxia through the inclusion of hypoxia-response element motifs in the expression cassette, the use of the anaerobic bacteria or the use of neutron irradiation. These results are encouraging and provide significant evidence that gene therapy targeted to the radiation field is a reasonably attractive therapeutic option and could help overcome hypoxic radioresistant tumors.

Impact of fractionation on out-of-field survival and DNA damage responses following exposure to intensity modulated radiation fields

To limit toxicity to normal tissues adjacent to the target tumour volume, radiotherapy is delivered using fractionated regimes whereby the total prescribed dose is given as a series of sequential smaller doses separated by specific time intervals. The impact of fractionation on out-of-field survival and DNA damage responses was determined in AGO-1522 primary human fibroblasts and MCF-7 breast tumour cells using uniform and modulated exposures delivered using a 225 kVp x-ray source. Responses to fractionated schedules (two equal fractions delivered with time intervals from 4 h to 48 h) were compared to those following acute exposures. Cell survival and DNA damage repair measurements indicate that cellular responses to fractionated non-uniform exposures differ from those seen in uniform exposures for the investigated cell lines. Specifically, there is a consistent lack of repair observed in the out-of-field populations during intervals between fractions, confirming the importance of cell signalling to out-of-field responses in a fractionated radiation schedule, and this needs to be confirmed for a wider range of cell lines and conditions.

Fractionated Radiation Exposure of Rat Spinal Cords Leads to Latent Neuro- Inflammation in Brain, Cognitive Deficits,and Alterations in Apurinic Endonuclease 1

Ionizing radiation causes degeneration of myelin, the insulating sheaths of neuronal axons, leading to neurological impairment. As radiation research on the central nervous system has predominantly focused on neurons, with few studies addressing the role of glial cells, we have focused our present research on identifying the latent effects of single/ fractionated -low dose of low/ high energy radiation on the role of base excision repair protein Apurinic Endonuclease-1, in the rat spinal cords oligodendrocyte progenitor cells’ differentiation. Apurinic endonuclease-1 is predominantly upregulated in response to oxidative stress by low- energy radiation, and previous studies show significant induction of Apurinic Endonuclease-1 in neurons and astrocytes. Our studies show for the first time, that fractionation of protons cause latent damage to spinal cord architecture while fractionation of HZE (28Si) induce increase in APE1 with single dose, which then decreased with fractionation. The oligodendrocyte progenitor cells differentiation was skewed with increase in immature oligodendrocytes and astrocytes, which likely cause the observed decrease in white matter, increased neuro-inflammation, together leading to the observed significant cognitive defects.

Investigating the Implications of a Variable RBE on Proton Dose Fractionation Across a Clinical Pencil Beam Scanned Spread-Out Bragg Peak

Purpose: To 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 Materials: Human 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.

Results: Significant 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.

Conclusions: The 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.

Gold nanoparticle induced vasculature damage in radiotherapy: Comparing protons, megavoltage photons, and kilovoltage photons

Purpose: The purpose of this work is to investigate the radiosensitizing effect of gold nanoparticle (GNP) induced vasculature damage for proton, megavoltage (MV) photon, and kilovoltage (kV) photon irradiation. Methods: Monte Carlo simulations were carried out using tool for particle simulation (TOPAS) to obtain the spatial dose distribution in close proximity up to 20 µm from the GNPs. The spatial dose distribution from GNPs was used as an input to calculate the dose deposited to the blood vessels. GNP induced vasculature damage was evaluated for three particle sources (a clinical spread out Bragg peak proton beam, a 6 MV photon beam, and two kV photon beams). For each particle source, various depths in tissue, GNP sizes (2, 10, and 20 nm diameter), and vessel diameters (8, 14, and 20 µm) were investigated. Two GNP distributions in lumen were considered, either homogeneously distributed in the vessel or attached to the inner wall of the vessel. Doses of 30 Gy and 2 Gy were considered, representing typical in vivo enhancement studies and conventional clinical fractionation, respectively. Results: These simulations showed that for 20 Au-mg/g GNP blood concentration homogeneously distributed in the vessel, the additional dose at the inner vascular wall encircling the lumen was 43% of the prescribed dose at the depth of treatment for the 250 kVp photon source, 1% for the 6 MV photon source, and 0.1% for the proton beam. For kV photons, GNPs caused 15% more dose in the vascular wall for 150 kVp source than for 250 kVp. For 6 MV photons, GNPs caused 0.2% more dose in the vascular wall at 20 cm depth in water as compared to at depth of maximum dose (Dmax). For proton therapy, GNPs caused the same dose in the vascular wall for all depths across the spread out Bragg peak with 12.7 cm range and 7 cm modulation. For the same weight of GNPs in the vessel, 2 nm diameter GNPs caused three times more damage to the vessel than 20 nm diameter GNPs. When the GNPs were attached to the inner vascular wall, the damage to the inner vascular wall can be up to 207% of the prescribed dose for the 250 kVp photon source, 4% for the 6 MV photon source, and 2% for the proton beam. Even though the average dose increase from the proton beam and MV photon beam was not large, there were high dose spikes that elevate the local dose of the parts of the blood vessel to be higher than 15 Gy even for 2 Gy prescribed dose, especially when the GNPs can be actively targeted to the endothelial cells. Conclusions: GNPs can potentially be used to enhance radiation therapy by causing vasculature damage through high dose spikes caused by the addition of GNPs especially for hypofractionated treatment. If GNPs are designed to actively accumulate at the tumor vasculature walls, vasculature damage can be increased significantly. The largest enhancement is seen using kilovoltage photons due to the photoelectric effect. Although no significant average dose enhancement was observed for the whole vasculature structure for both MV photons and protons, they can cause high local dose escalation (>15 Gy) to areas of the blood vessel that can potentially contribute to the disruption of the functionality of the blood vessels in the tumor.

Roadmap to clinical use of gold nanoparticles for radiation sensitization

The past decade has seen a dramatic increase in interest in the use of gold nanoparticles (GNPs) as radiation sensitizers for radiation therapy. This interest was initially driven by their strong absorption of ionizing radiation and the resulting ability to increase dose deposited within target volumes even at relatively low concentrations. These early observations are supported by extensive experimental validation, showing GNPs' efficacy at sensitizing tumors in both in vitro and in vivo systems to a range of types of ionizing radiation, including kilovoltage and megavoltage X rays as well as charged particles. Despite this experimental validation, there has been limited translation of GNP-mediated radiation sensitization to a clinical setting. One of the key challenges in this area is the wide range of experimental systems that have been investigated, spanning a range of particle sizes, shapes, and preparations. As a result, mechanisms of uptake and radiation sensitization have remained difficult to clearly identify. This has proven a significant impediment to the identification of optimal GNP formulations which strike a balance among their radiation sensitizing properties, their specificity to the tumors, their biocompatibility, and their imageability in vivo. This white paper reviews the current state of knowledge in each of the areas concerning the use of GNPs as radiosensitizers, and outlines the steps which will be required to advance GNP-enhanced radiation therapy from their current pre-clinical setting to clinical trials and eventual routine usage.

Conventional versus hypofractionated high-dose intensity-modulated radiotherapy for prostate cancer: 5-year outcomes of the randomised, non-inferiority, phase 3 CHHiP trial

BACKGROUND: Prostate cancer might have high radiation-fraction sensitivity that would give a therapeutic advantage to hypofractionated treatment. We present a pre-planned analysis of the efficacy and side-effects of a randomised trial comparing conventional and hypofractionated radiotherapy after 5 years follow-up.

METHODS: CHHiP is a randomised, phase 3, non-inferiority trial that recruited men with localised prostate cancer (pT1b-T3aN0M0). Patients were randomly assigned (1:1:1) to conventional (74 Gy delivered in 37 fractions over 7·4 weeks) or one of two hypofractionated schedules (60 Gy in 20 fractions over 4 weeks or 57 Gy in 19 fractions over 3·8 weeks) all delivered with intensity-modulated techniques. Most patients were given radiotherapy with 3-6 months of neoadjuvant and concurrent androgen suppression. Randomisation was by computer-generated random permuted blocks, stratified by National Comprehensive Cancer Network (NCCN) risk group and radiotherapy treatment centre, and treatment allocation was not masked. The primary endpoint was time to biochemical or clinical failure; the critical hazard ratio (HR) for non-inferiority was 1·208. Analysis was by intention to treat. Long-term follow-up continues. The CHHiP trial is registered as an International Standard Randomised Controlled Trial, number ISRCTN97182923.

FINDINGS: Between Oct 18, 2002, and June 17, 2011, 3216 men were enrolled from 71 centres and randomly assigned (74 Gy group, 1065 patients; 60 Gy group, 1074 patients; 57 Gy group, 1077 patients). Median follow-up was 62·4 months (IQR 53·9-77·0). The proportion of patients who were biochemical or clinical failure free at 5 years was 88·3% (95% CI 86·0-90·2) in the 74 Gy group, 90·6% (88·5-92·3) in the 60 Gy group, and 85·9% (83·4-88·0) in the 57 Gy group. 60 Gy was non-inferior to 74 Gy (HR 0·84 [90% CI 0·68-1·03], pNI=0·0018) but non-inferiority could not be claimed for 57 Gy compared with 74 Gy (HR 1·20 [0·99-1·46], pNI=0·48). Long-term side-effects were similar in the hypofractionated groups compared with the conventional group. There were no significant differences in either the proportion or cumulative incidence of side-effects 5 years after treatment using three clinician-reported as well as patient-reported outcome measures. The estimated cumulative 5 year incidence of Radiation Therapy Oncology Group (RTOG) grade 2 or worse bowel and bladder adverse events was 13·7% (111 events) and 9·1% (66 events) in the 74 Gy group, 11·9% (105 events) and 11·7% (88 events) in the 60 Gy group, 11·3% (95 events) and 6·6% (57 events) in the 57 Gy group, respectively. No treatment-related deaths were reported.

INTERPRETATION: Hypofractionated radiotherapy using 60 Gy in 20 fractions is non-inferior to conventional fractionation using 74 Gy in 37 fractions and is recommended as a new standard of care for external-beam radiotherapy of localised prostate cancer.

FUNDING: Cancer Research UK, Department of Health, and the National Institute for Health Research Cancer Research Network.