Developments in the genome organisation field has resulted in the recent methodology to infer spatial conformations of the genome directly from experimentally measured genome contacts (Hi-C data). ...This provides a detailed description of both intra- and inter-chromosomal arrangements. Chromosomal intermingling is an important driver for radiation-induced DNA mis-repair. Which is a key biological endpoint of relevance to the fields of cancer therapy (radiotherapy), public health (biodosimetry) and space travel. For the first time, we leverage these methods of inferring genome organisation and couple them to nano-dosimetric radiation track structure modelling to predict quantities and distribution of DNA damage within cell-type specific geometries. These nano-dosimetric simulations are highly dependent on geometry and are benefited from the inclusion of experimentally driven chromosome conformations. We show how the changes in Hi-C contract maps impact the inferred geometries resulting in significant differences in chromosomal intermingling. We demonstrate how these differences propagate through to significant changes in the distribution of DNA damage throughout the cell nucleus, suggesting implications for DNA repair fidelity and subsequent cell fate. We suggest that differences in the geometric clustering for the chromosomes between the cell-types are a plausible factor leading to changes in cellular radiosensitivity. Furthermore, we investigate changes in cell shape, such as flattening, and show that this greatly impacts the distribution of DNA damage. This should be considered when comparing in vitro results to in vivo systems. The effect may be especially important when attempting to translate radiosensitivity measurements at the experimental in vitro level to the patient or human level.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Preclinical radiation research lacks standardized dosimetry procedures that provide traceability to a primary standard. Consequently, ensuring accuracy and reproducibility between studies is ...challenging. Using 3D printed murine phantoms we undertook a dosimetry audit of Xstrahl Small Animal Radiation Research Platforms (SARRPs) installed at 7 UK centres. The geometrically realistic phantom accommodated alanine pellets and Gafchromic EBT3 film for simultaneous measurement of the dose delivered and the dose distribution within a 2D plane, respectively. Two irradiation scenarios were developed: (1) a 10 × 10 mm
static field targeting the pelvis, and (2) a 5 × 5 mm
90° arc targeting the brain. For static fields, the absolute difference between the planned dose and alanine measurement across all centres was 4.1 ± 4.3% (mean ± standard deviation), with an overall range of - 2.3 to 10.5%. For arc fields, the difference was - 1.2% ± 6.1%, with a range of - 13.1 to 7.7%. EBT3 dose measurements were greater than alanine by 2.0 ± 2.5% and 3.5 ± 6.0% (mean ± standard deviation) for the static and arc fields, respectively. 2D dose distributions showed discrepancies to the planned dose at the field edges. The audit demonstrates that further work on preclinical radiotherapy quality assurance processes is merited.
The UK has an important role in the evaluation of proton beam therapy (PBT) and takes its place on the world stage with the opening of the first National Health Service (NHS) PBT centre in Manchester ...in 2018, and the second in London coming in 2020. Systematic evaluation of the role of PBT is a key objective. By September 2019, 108 patients had started treatment, 60 paediatric, 19 teenagers and young adults and 29 adults. Obtaining robust outcome data is vital, if we are to understand the strengths and weaknesses of current treatment approaches. This is important in demonstrating when PBT will provide an advantage and when it will not, and in quantifying the magnitude of benefit.The UK also has an important part to play in translational PBT research, and building a research capability has always been the vision. We are perfectly placed to perform translational pre-clinical biological and physical experiments in the dedicated research room in Manchester. The nature of DNA damage from proton irradiation is considerably different from X-rays and this needs to be more fully explored. A better understanding is needed of the relative biological effectiveness (RBE) of protons, especially at the end of the Bragg peak, and of the effects on tumour and normal tissue of PBT combined with conventional chemotherapy, targeted drugs and immunomodulatory agents. These experiments can be enhanced by deterministic mathematical models of the molecular and cellular processes of DNA damage response. The fashion of ultra-high dose rate FLASH irradiation also needs to be explored.
The process of end-joining during nonhomologous repair of DNA double-strand breaks (DSBs) after radiation damage is considered. Experimental evidence has revealed that the dynamics of DSB ends ...exhibit subdiffusive motion rather than simple diffusion with rare directional movement. Traditional models often overlook the rare long-range directed motion. To address this limitation, we present a heterogeneous anomalous diffusion model consisting of subdiffusive fractional Brownian motion interchanged with short periods of long-range movement. Our model sheds light on the underlying mechanisms of heterogeneous diffusion in DSB repair and could be used to quantify the DSB dynamics on a time scale inaccessible to single particle tracking analysis. The model predicts that the long-range movement of DSB ends is responsible for the misrepair of DSBs in the form of dicentric chromosome lesions.
The cytotoxicity of radiotherapy and chemotherapy can be enhanced by modulating DNA repair. PARP is a family of enzymes required for an efficient base-excision repair of DNA single-strand breaks and ...inhibition of PARP can prevent the repair of these lesions. The current study investigates the trimodal combination of ABT-888, a potent inhibitor of PARP1-2, ionizing radiation and temozolomide(TMZ)-based chemotherapy in glioblastoma (GBM) cells.
Four human GBM cell lines were treated for 5 h with 5 μM ABT-888 before being exposed to X-rays concurrently with TMZ at doses of 5 or 10 μM for 2 h. ABT-888's PARP inhibition was measured using immunodetection of poly(ADP-ribose) (pADPr). Cell survival and the different cell death pathways were examined via clonogenic assay and morphological characterization of the cell and cell nucleus.
Combining ABT-888 with radiation yielded enhanced cell killing in all four cell lines, as demonstrated by a sensitizer enhancement ratio at 50% survival (SER50) ranging between 1.12 and 1.37. Radio- and chemo-sensitization was further enhanced when ABT-888 was combined with both X-rays and TMZ in the O6-methylguanine-DNA-methyltransferase (MGMT)-methylated cell lines with a SER50 up to 1.44. This effect was also measured in one of the MGMT-unmethylated cell lines with a SER50 value of 1.30. Apoptosis induction by ABT-888, TMZ and X-rays was also considered and the effect of ABT-888 on the number of apoptotic cells was noticeable at later time points. In addition, this work showed that ABT-888 mediated sensitization is replication dependent, thus demonstrating that this effect might be more pronounced in tumour cells in which endogenous replication lesions are present in a larger proportion than in normal cells.
This study suggests that ABT-888 has the clinical potential to enhance the current standard treatment for GBM, in combination with conventional chemo-radiotherapy. Interestingly, our results suggest that the use of PARP inhibitors might be clinically significant in those patients whose tumour is MGMT-unmethylated and currently derive less benefit from TMZ.
Abstract
Micronucleus (MN) formation is routinely used as a biodosimeter for radiation exposures and has historically been used as a measure of DNA damage in cells. Strongly correlating with dose, MN ...are also suggested to indicate radiation quality, differentiating between particle and photon irradiation. The “gold standard” for measuring MN formation is Fenech’s cytokinesis-block micronucleus (CBMN) cytome assay, which uses the cytokinesis blocking agent cytochalasin-B. Here, we present a comprehensive analysis of the literature investigating MN induction trends in vitro, collating 193 publications, with 2476 data points. Data were collected from original studies that used the CBMN assay to quantify MN in response to ionizing radiation in vitro. Overall, the meta-analysis showed that individual studies mostly have a linear increase of MN with dose 85% of MN per cell (MNPC) datasets and 89% of percentage containing MN (PCMN) datasets had an R2 greater than 0.90. However, there is high variation between studies, resulting in a low R2 when data are combined (0.47 for MNPC datasets and 0.60 for PCMN datasets). Particle type, species, cell type, and cytochalasin-B concentration were suggested to influence MN frequency. However, variation in the data meant that the effects could not be strongly correlated with the experimental parameters investigated. There is less variation between studies when comparing the PCMN rather than the number of MNPC. Deviation from CBMN protocol specified timings did not have a large effect on MN induction. However, further analysis showed less variation between studies following Fenech’s protocol closely, which provided more reliable results. By limiting the cell type and species as well as only selecting studies following the Fenech protocol, R2 was increased to 0.64 for both measures. We therefore determine that due to variation between studies, MN are currently a poor predictor of radiation-induced DNA damage and make recommendations for futures studies assessing MN to improve consistency between datasets.
Preclinical radiotherapy studies using small animals are an indispensable step in the pathway from in vitro experiments to clinical implementation. As radiotherapy techniques advance in the clinic, ...it is important that preclinical models evolve to keep in line with these developments. The use of orthotopic tumour sites, the development of tissue-equivalent mice phantoms and the recent introduction of image-guided small animal radiation research platforms has enabled similar precision treatments to be delivered in the laboratory.These technological developments, however, are hindered by a lack of corresponding dosimetry standards and poor reporting of methodologies. Without robust and well documented preclinical radiotherapy quality assurance processes, it is not possible to ensure the accuracy and repeatability of dose measurements between laboratories. As a consequence current RT-based preclinical models are at risk of becoming irrelevant.In this review we explore current standardization initiatives, focusing in particular on recent developments in small animal irradiation equipment, 3D printing technology to create customisable tissue-equivalent dosimetry phantoms and combining these phantoms with commonly used detectors.
Gold nanoparticles have been proven as potential radiosensitizer when combined with protons. Initially the radiosensitization effect was attributed to the physical interactions of radiation with the ...gold and the production of secondary electrons that induce DNA damage. However, emerging data challenge this hypothesis, supporting the existence of alternative or supplementary radiosensitization mechanisms. In this work we incorporate a realistic cell model with detailed DNA geometry and a realistic gold nanoparticle biodistribution based on experimental data. The DNA single and double strand breaks, and damage complexity are counted under various scenarios of different gold nanoparticle size, biodistribution and concentration, and proton energy. The locality of the effect, i.e. the existence of higher damage at a location close to the gold distribution, is also addressed by investigating the DNA damage at a chromosomal territory. In all the cases we do not observe any significant increase in the single/double strand break yield or damage complexity in the presence of gold nanoparticles under proton irradiation; nor there is a locality to the effect. Our results show for the first time that the physical interactions of protons with the gold nanoparticles should not be considered directly responsible for the observed radiosensitization effect. The model used only accounts for DNA damage from direct interactions, whilst considering the indirect effect, and it is possible the radiosensitization effect to be due to other physical effects, although we consider that possibility unlikely. Our conclusion suggests that other mechanisms might have greater contribution to the radiosensitization effect and further investigation should be conducted.
High-energy Proton Beam Therapy (PBT) commenced in England in 2018 and NHS England commissions PBT for 1.5% of patients receiving radical radiotherapy. We sought expert opinion on the level of ...provision.
Invitations were sent to 41 colleagues working in PBT, most at one UK centre, to contribute by completing a spreadsheet. 39 responded: 23 (59%) completed the spreadsheet; 16 (41%) declined, arguing that clinical outcome data are lacking, but joined six additional site-specialist oncologists for two consensus meetings. The spreadsheet was pre-populated with incidence data from Cancer Research UK and radiotherapy use data from the National Cancer Registration and Analysis Service. 'Mechanisms of Benefit' of reduced growth impairment, reduced toxicity, dose escalation and reduced second cancer risk were examined.
The most reliable figure for percentage of radical radiotherapy patients likely to benefit from PBT was that agreed by 95% of the 23 respondents at 4.3%, slightly larger than current provision. The median was 15% (range 4-92%) and consensus median 13%. The biggest estimated potential benefit was from reducing toxicity, median benefit to 15% (range 4-92%), followed by dose escalation median 3% (range 0 to 47%); consensus values were 12 and 3%. Reduced growth impairment and reduced second cancer risk were calculated to benefit 0.5% and 0.1%.
The most secure estimate of percentage benefit was 4.3% but insufficient clinical outcome data exist for confident estimates. The study supports the NHS approach of using the evidence base and developing it through randomised trials, non-randomised studies and outcomes tracking.
Less is known about the percentage of patients who may benefit from PBT than is generally acknowledged. Expert opinion varies widely. Insufficient clinical outcome data exist to provide robust estimates. Considerable further work is needed to address this, including international collaboration; much is already underway but will take time to provide mature data.
To investigate and benchmark the current clinical and dosimetric practices in stereotactic radiosurgery (SRS) in the UK.
A detailed questionnaire was sent to 70 radiotherapy centres in the UK. 97% ...(68/70) of centres replied between June and December 2014.
21 centres stated that they are practising SRS, and a further 12 centres plan to start SRS by the end of 2016. The most commonly treated indications are brain metastases and acoustic neuromas. A large range of prescription isodoses that range from 45% to 100% between different radiotherapy centres was seen. Ionization chambers and solid-water phantoms are used by the majority of centres for patient-specific quality assurance, and thermoplastic masks for patient immobilization are more commonly used than fixed stereotactic frames. The majority of centres perform orthogonal kilovoltage X-rays for localization before and during delivery. The acceptable setup accuracy reported ranges from 0.1 to 2 mm with a mean of 0.8 mm.
SRS has been increasing in use in the UK and will continue to increase in the next 2 years. There is no current consensus between SRS centres as a whole, or even between SRS centres with the same equipment, on the practices followed. This indicates the need for benchmarking and standardization in SRS practices within the UK.
This article outlines the current practices in SRS and provides a benchmark for reference and comparison with future research in this technique.
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