First independent validation of the proton-boron capture therapy concept.

Boron has been suggested to enhance the biological effectiveness of proton beams in the Bragg peak region via the p + 11B → 3α nuclear capture reaction. However, a number of groups have observed no such enhancement in vitro or questioned its proposed mechanism recently. To help elucidate this phenomenon, we irradiated DU145 prostate cancer or U-87 MG glioblastoma cells by clinical 190 MeV proton beams in plateau or Bragg peak regions with or without 10B or 11B isotopes added as sodium mercaptododecaborate (BSH). The results demonstrate that 11B but not 10B or other components of the BSH molecule enhance cell killing by proton beams. The enhancement occurs selectively in the Bragg peak region, is present for boron concentrations as low as 40 ppm, and is not due to secondary neutrons. The enhancement is likely initiated by proton-boron capture reactions producing three alpha particles, which are rare events occurring in a few cells only, and their effects are amplified by intercellular communication to a population-level response. The observed up to 2-3-fold reductions in survival levels upon the presence of boron for the studied prostate cancer or glioblastoma cells suggest promising clinical applications for these tumour types.

Effect of elevated temperature and hydrocortisone addition on the proliferation of fibroblasts.

Hyperthermia along with hydrocortisone (HC) are proven teratogens that can negatively influence embryo development during early pregnancy. Proliferation of cells is one of the main developmental processes during the early embryogenesis. This study was focused on testing the effect of elevated temperature and HC addition on proliferation of cells in in vitro cultures. The V79-4 cell line was treated with HC and cultured in vitro at 37 °C or 39 °C, respectively. To reveal the effect of both factors, the proliferation of cells cultured under different conditions was evaluated using various approaches (colony formation assay, generation of growth curves, computation of doubling times, and mitotic index estimation). Our results indicate that a short-term exposure to elevated temperature slightly stimulates and a long-term exposure suppresses cell proliferation. However, HC (0.1 mg/ml) acts as a stimulator of cell proliferation. Interestingly, the interaction of HC and long-term elevated temperature (39 °C) exposure results in at least partial compensation of the negative impact of elevated temperature by HC addition and in higher proliferation if compared with cells cultured at 39 °C without addition of HC.

Out-of-Field Doses Produced by a Proton Scanning Beam Inside Pediatric Anthropomorphic Phantoms and Their Comparison With Different Photon Modalities.

Since 2010, EURADOS Working Group 9 (Radiation Dosimetry in Radiotherapy) has been involved in the investigation of secondary and scattered radiation doses in X-ray and proton therapy, especially in the case of pediatric patients. The main goal of this paper is to analyze and compare out-of-field neutron and non-neutron organ doses inside 5- and 10-year-old pediatric anthropomorphic phantoms for the treatment of a 5-cm-diameter brain tumor. Proton irradiations were carried out at the Cyclotron Centre Bronowice in IFJ PAN Krakow Poland using a pencil beam scanning technique (PBS) at a gantry with a dedicated scanning nozzle (IBA Proton Therapy System, Proteus 235). Thermoluminescent and radiophotoluminescent dosimeters were used for non-neutron dose measurements while secondary neutrons were measured with track-etched detectors. Out-of-field doses measured using intensity-modulated proton therapy (IMPT) were compared with previous measurements performed within a WG9 for three different photon radiotherapy techniques: 1) intensity-modulated radiation therapy (IMRT), 2) three-dimensional conformal radiation therapy (3D CDRT) performed on a Varian Clinac 2300 linear accelerator (LINAC) in the Centre of Oncology, Krakow, Poland, and 3) Gamma Knife surgery performed on the Leksell Gamma Knife (GK) at the University Hospital Centre Zagreb, Croatia. Phantoms and detectors used in experiments as well as the target location were the same for both photon and proton modalities. The total organ dose equivalent expressed as the sum of neutron and non-neutron components in IMPT was found to be significantly lower (two to three orders of magnitude) in comparison with the different photon radiotherapy techniques for the same delivered tumor dose. For IMPT, neutron doses are lower than non-neutron doses close to the target but become larger than non-neutron doses further away from the target. Results of WG9 studies have provided out-of-field dose levels required for an extensive set of radiotherapy techniques, including proton therapy, and involving a complete description of organ doses of pediatric patients. Such studies are needed for validating mathematical models and Monte Carlo simulation tools for out-of-field dosimetry which is essential for dedicated epidemiological studies which evaluate the risk of second cancers and other late effects for pediatric patients treated with radiotherapy.

BORON-ENHANCED BIOLOGICAL EFFECTIVENESS OF PROTON IRRADIATION: STRATEGY TO ASSESS THE UNDERPINNING MECHANISM.

Proton radiotherapy for the treatment of cancer offers an excellent dose distribution. Cellular experiments have shown that in terms of biological effects, the sharp dose distribution is further amplified, by as much as 75%, in the presence of boron. It is a matter of debate whether the underlying physical processes involve the nuclear reaction of 11B with protons or 10B with secondary neutrons, both producing densely ionizing short-ranged particles. Likewise, potential roles of intercellular communication or boron acting as a radiosensitizer are not clear. We present an ongoing research project based on a multiscale approach to elucidate the mechanism by which boron enhances the effectiveness of proton irradiation in the Bragg peak. It combines experimental with simulation tools to study the physics of proton-boron interactions, and to analyze intra- and inter-cellular boron biology upon proton irradiation.

MONTE CARLO SIMULATIONS OF OUT-OF-FIELD LET SPECTRA IN WATER PHANTOM IRRADIATED BY SCANNING PENCIL PROTON BEAM.

LET spectra can be measured by track-etched detectors. However, these detectors are not able to identify the type of interacting particles. Monte Carlo simulations can provide this missing information. In this work, Monte Carlo simulations based on the EURADOS Work Group 9 experiment consisting of systematic 3D mapping of out-of-field doses and LET spectra in a prototype water phantom were performed. The simulations aimed to identify the types of particles contributing to the out-of-field LET spectra. The total absorbed dose, LET and energy spectra were calculated. The calculated dose distributions and LET spectra were compared with the ones measured by radiophotoluminiscence and track-etched detectors. The out-of-field particles and their LET values were identified. No statistically significant differences between the measured and simulated spectra were revealed in the LET range of 100-2000 keV µm-1.

EXPERIMENTAL IN VITRO DOSIMETRY OF 223RA AND 177LU.

Targeted alpha therapy with radionuclides undergoing multiple alpha-particle decays is a promising method of nuclear medicine. To study the effectiveness of alpha versus beta emitters, survival of DU145 prostate cancer cells exposed to 223Ra or 177Lu was assessed. Per decay, the cells were much more sensitive to the alpha than beta emitter. However, per unit dose the sensitivities would be comparable, contrary to the well-known evidence, if the decay energy were deposited within the sample completely and homogeneously. Measurements by Timepix detectors showed about three times higher counts of alpha particles above than below the sample. After the first alpha decay of 223Ra to 219Rn, this gas likely moves upwards and its subsequent three alpha decays occur in the upper part of the sample. Correct estimation of absorbed dose is a critical issue when analysing in vitro data and when translating their results to clinical applications.

RADIATION DAMAGE TO DNA PLASMIDS IN THE PRESENCE OF BOROCAPTATES.

Boron derivatives have great potential in cancer diagnostics and treatment. Borocaptates are used in boron neutron capture therapy and potentially in proton boron fusion therapy. This work examines modulation effects of two borocaptate compounds on radiation-induced DNA damage. Aqueous solutions of pBR322 plasmid containing increasing concentrations of borocaptates were irradiated with 60Co gamma rays or 30 MeV protons. Induction of single and double DNA strand breaks was investigated using agarose gel electrophoresis. In this model system, representing DNA without the intervention of cellular repair mechanisms, the boron derivatives acted as antioxidants. Clinically relevant boron concentrations of 40 ppm reduced the DNA single strand breakage seven-fold. Possible mechanisms of the observed effect are discussed.

Out-of-field doses in pediatric craniospinal irradiations with 3D-CRT, VMAT, and scanning proton radiotherapy: A phantom study.

PURPOSE: Craniospinal irradiation (CSI) has greatly increased survival rates for patients with a diagnosis of medulloblastoma and other primitive neuroectodermal tumors. However, as it includes exposure of a large volume of healthy tissue to unwanted doses, there is a strong concern about the complications of the treatment, especially for the children. To estimate the risk of second cancers and other unwanted effects, out-of-field dose assessment is necessary. The purpose of this study is to evaluate and compare out-of-field doses in pediatric CSI treatment using conventional and advanced photon radiotherapy (RT) and advanced proton therapy. To our knowledge, it is the first such comparison based on in-phantom measurements. Additionally, for out-of-field doses during photon RT in this and other studies, comparisons were made using analytical modeling. METHODS: In order to describe the out-of-field doses absorbed in a pediatric patient during actual clinical treatment, an anthropomorphic phantom, which mimics the 10-year-old child, was used. Photon 3D-conformal RT (3D-CRT) and two advanced, highly conformal techniques: photon volumetric-modulated arc therapy (VMAT) and active pencil beam scanning (PBS) proton RT were used for CSI treatment. Radiophotoluminescent and poly-allyl-diglycol-carbonate nuclear track detectors were used for photon and neutron dosimetry in the phantom, respectively. Out-of-field doses from neutrons were expressed in terms of dose equivalent. A two-Gaussian model was implemented for out-of-field doses during photon RT. RESULTS: The mean VMAT photon doses per target dose to all organs in this study were under 50% of the target dose (i.e., <500 mGy/Gy), while the mean 3D-CRT photon dose to oesophagus, gall bladder, and thyroid, exceeded that value. However, for 3D-CRT, better sparing was achieved for eyes and lungs. The mean PBS photon doses for all organs were up to three orders of magnitude lower compared to VMAT and 3D-CRT and exceeded 10 mGy/Gy only for the oesophagus, intestine, and lungs. The mean neutron dose equivalent during PBS for eight organs of interest (thyroid, breasts, lungs, liver, stomach, gall bladder, bladder, prostate) ranged from 1.2 mSv/Gy for bladder to 23.1 mSv/Gy for breasts. Comparison of out-of-field doses in this and other phantom studies found in the literature showed that a simple and fast two-Gaussian model for out-of-field doses as a function of distance from the field edge can be applied in a CSI using photon RT techniques. CONCLUSIONS: PBS is the most promising technique for out-of-field dose reduction in comparison to photon techniques. Among photon techniques, VMAT is a preferred choice for most of out-of-field organs and especially for the thyroid, while doses for eyes, breasts, and lungs are lower for 3D-CRT. For organs outside the field edge, a simple analytical model can be helpful for clinicians involved in treatment planning using photon RT but also for retrospective data analysis for cancer risk estimates and epidemiology in general.

Folding DNA into origami nanostructures enhances resistance to ionizing radiation.

We report experimental results on damage induced by ionizing radiation to DNA origami triangles which are commonly used prototypes for scaffolded DNA origami nanostructures. We demonstrate extreme stability of DNA origami upon irradiation, which is caused by (i) the multi-row design holding the shape of the origami even after severe damage to the scaffold DNA and (ii) the reduction of damage to the scaffold DNA due to the protective effect of the folded structure. With respect to damage induced by ionizing radiation, the protective effect of the structure is superior to that of a naturally paired DNA double helix. Present results allow estimating the stability of scaffolded DNA origami nanostructures in applications such as nanotechnology, pharmacy or in singulo molecular studies where they are exposed to ionizing radiation from natural and artificial sources. Additionally, possibilities are opened for scaffolded DNA use in the design of radiation-resistant and radio-sensitive materials.

Gamma radiation effects on diamond field-effect biosensors with fibroblasts and extracellular matrix.

Due to high biocompatibility, miniaturization, optical transparency and low production cost together with high radiation hardness the diamond-based sensors are considered promising for radiation medicine and biomedicine in general. Here we present detection of fibroblast cell culture properties by nanocrystalline diamond solution-gated field-effect transistors (SG-FET), including effects of gamma irradiation. We show that blank nanocrystalline diamond field-effect biosensors are stable at least up to 300 Gy of γ irradiation. On the other hand, gate current of the diamond SG-FET biosensors with fibroblastic cells increases exponentially over an order of magnitude with increasing radiation dose. Extracellular matrix (ECM) formation is also detected and analyzed by correlation of electronic sensor data with optical, atomic force, fluorescence, and scanning electron microscopies.

Assessment of DNA damage with an adapted independent reaction time approach implemented in Geant4-DNA for the simulation of diffusion-controlled reactions between radio-induced reactive species and a chromatin fiber.

PURPOSE: Simulation of indirect damage originating from the attack of free radical species produced by ionizing radiation on biological molecules based on the independent pair approximation is investigated in this work. In addition, a new approach, relying on the independent pair approximation that is at the origin of the independent reaction time (IRT) method, is proposed in the chemical stage of Geant4-DNA. METHODS: This new approach has been designed to respect the current Geant4-DNA chemistry framework while proposing a variant IRT method. Based on the synchronous algorithm, this implementation allows us to access the information concerning the position of radicals and may make it more convenient for biological damage simulations. Estimates of the evolution of free species as well as biological hits in a segment of DNA chromatin fiber in Geant4-DNA were compared for the dynamic time step approach of the step-by-step (SBS) method, currently used in Geant4-DNA, and this newly implemented IRT. RESULTS: Results show a gain in computation time of a factor of 30 for high LET particle tracks with a better than 10% agreement on the number of DNA hits between the value obtained with the IRT method as implemented in this work and the SBS method currently available in Geant4-DNA. CONCLUSION: Offering in Geant4-DNA more efficient methods for the chemical step based on the IRT method is a task in progress. For the calculation of biological damage, information on the position of chemical species is a crucial point. This can be achieved using the method presented in this paper.

In Vivo Validation of Alternative FDXR Transcripts in Human Blood in Response to Ionizing Radiation.

Following cell stress such as ionising radiation (IR) exposure, multiple cellular pathways are activated. We recently demonstrated that ferredoxin reductase (FDXR) has a remarkable IR-induced transcriptional responsiveness in blood. Here, we provided a first comprehensive FDXR variant profile following DNA damage. First, specific quantitative real-time polymerase chain reaction (qPCR) primers were designed to establish dose-responses for eight curated FDXR variants, all up-regulated after IR in a dose-dependent manner. The potential role of gender on the expression of these variants was tested, and neither the variants response to IR nor the background level of expression was profoundly affected; moreover, in vitro induction of inflammation temporarily counteracted IR response early after exposure. Importantly, transcriptional up-regulation of these variants was further confirmed in vivo in blood of radiotherapy patients. Full-length nanopore sequencing was performed to identify other FDXR variants and revealed the high responsiveness of FDXR-201 and FDXR-208. Moreover, FDXR-218 and FDXR-219 showed no detectable endogenous expression, but a clear detection after IR. Overall, we characterised 14 FDXR transcript variants and identified for the first time their response to DNA damage in vivo. Future studies are required to unravel the function of these splicing variants, but they already represent a new class of radiation exposure biomarkers.

In Vitro Comparison of Passive and Active Clinical Proton Beams.

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.

Overcoming challenges in human saliva gene expression measurements.

Saliva, as a non-invasive and easily accessible biofluid, has been shown to contain RNA biomarkers for prediction and diagnosis of several diseases. However, systematic analysis done by our group identified two problematic issues not coherently described before: (1) most of the isolated RNA originates from the oral microbiome and (2) the amount of isolated human RNA is comparatively low. The degree of bacterial contamination showed ratios up to 1:900,000, so that only about one out of 900,000 RNA copies was of human origin, but the RNA quality (average RIN 6.7 + /- 0.8) allowed for qRT-PCR. Using 12 saliva samples from healthy donors, we modified the methodology to (1) select only human RNA during cDNA synthesis by aiming at the poly(A)+-tail and (2) introduced a pre-amplification of human RNA before qRT-PCR. Further, the manufacturer's criteria for successful pre-amplification (Ct values ≤ 35 for unamplified cDNA) had to be replaced by (3) proofing linear pre-amplification for each gene, thus, increasing the number of evaluable samples up to 70.6%. When considering theses three modifications unbiased gene expression analysis on human salivary RNA can be performed.

RADIOPROTECTIVE EFFECT OF HYDROXYL RADICAL SCAVENGERS ON PROKARYOTIC AND EUKARYOTIC CELLS UNDER VARIOUS GAMMA IRRADIATION CONDITIONS.

The influence of various hydroxyl radical scavengers such as methanol, ethanol and dimethyl sulfoxide on radiation sensitivity of prokaryotic cells (bacteria Escherichia coli) and eukaryotic cells (yeast Saccharomyces cerevisiae and V79 cells-Chinese hamster pulmonary fibroblasts) irradiated by 60Co gamma radiation was investigated. The dependence of radiation sensitivity on dose rate in range from 1.8 to 100 Gy h-1 was evaluated. Survival of cells irradiated by increasing dose rates was followed using clonogenic assay. Specific protective effect was found to be a nonmonotonous function of dose rate with typical maximum at the dose rate range from 50 to 55 Gy h-1 in all studied cell types.

COMPARISON OF THE RADIATION SENSITIVITY OF THE BREAST CANCER CELL LINE MCF7 AND HUMAN ADIPOSE-DERIVED STEM CELLS.

A comparison between breast cancer cell line MCF7 and human adipose-derived stem cells (ADSC) after irradiation by the same doses of megavoltage X-rays was performed. The cell growth, the induction of apoptosis and the expression of selected genes were analyzed. Irradiated MCF7 related to its control sample grows slower than ADSC and it undergoes apoptosis in much higher levels than ADSC. This was confirmed by real-time polymerase chain reaction as well, where the expression of apoptotic genes was found to be considerably higher for MCF7 than for ADSC. From the results of this project, it could be stated that MCF7 is more radiosensitive than ADSC.

RADIATION-INDUCED PLASMID DNA DAMAGE: EFFECT OF CONCENTRATION AND LENGTH.

Plasmid DNA is commonly used as a simpler substitute for a cell in studies of early effects of ionizing radiation because it allows to determine yields of primary DNA lesions. Experimental studies often employ plasmids of different lengths, in different concentrations in the aqueous solution. Influence of these parameters on the heavy-ion induced yields of primary DNA damage has been studied, using plasmids pUC19 (2686 bp), pBR322 (4361 bp) and pKLAC2 (9107 bp) in 10 and 50 ng/µl concentration. Results demonstrate the impact of plasmid length, while no significant difference was observed between the two concentrations. The uncertainty of the results is discussed.

OVERVIEW OF AIRCREW EXPOSURE TO COSMIC RADIATION IN THE CZECH REPUBLIC.

Monitoring of exposures of aircrew members of airline operators registered in the Czech Republic has been performed since 1998. The individual effective doses are calculated using the computer code CARI annually. The calculations are based on information about participation of aircrew members on the specific flights and on the parameters describing the typical flight profiles of the flights; the latter is regularly verified with control measurements performed onboard aircraft. The results show that (1) the average annual effective doses in the period from 1998 to 2017 range from 1.3 to 2.1 mSv, (2) the maximum effective dose of a crew member is 5.7 mSv and (3) the annual collective effective doses range from 1.3 to 4.1 manSv. Combined relative uncertainty of the results is ~25%.

Differentiation Induction as a Response to Irradiation in Neural Stem Cells In Vitro.

Radiotherapy plays a significant role in brain cancer treatment; however, the use of this therapy is often accompanied by neurocognitive decline that is, at least partially, a consequence of radiation-induced damage to neural stem cell populations. Our findings describe features that define the response of neural stem cells (NSCs) to ionizing radiation. We investigated the effects of irradiation on neural stem cells isolated from the ventricular-subventricular zone of mouse brain and cultivated in vitro. Our findings describe the increased transcriptional activity of p53 targets and proliferative arrest after irradiation. Moreover, we show that most cells do not undergo apoptosis after irradiation but rather cease proliferation and start a differentiation program. Induction of differentiation and the demonstrated potential of irradiated cells to differentiate into neurons may represent a mechanism whereby damaged NSCs eliminate potentially hazardous cells and circumvent the debilitating consequences of cumulative DNA damage.