Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability.

Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery. In response to DNA damage, this complex regulates pre-mRNA splicing of a number of genes involved in DNA damage signaling and repair, thereby promoting the stability of these transcripts/proteins. Further, we show that abrogation of this complex results in sensitivity to DNA damage, defective DNA repair, and genomic instability. Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types. These data suggest that regulation of splicing by the BRCA1-mRNA splicing complex plays an important role in the cellular response to DNA damage.

Relationship of In Vitro Toxicity of Technetium-99m to Subcellular Localisation and Absorbed Dose.

Auger electron-emitters increasingly attract attention as potential radionuclides for molecular radionuclide therapy in oncology. The radionuclide technetium-99m is widely used for imaging; however, its potential as a therapeutic radionuclide has not yet been fully assessed. We used MDA-MB-231 breast cancer cells engineered to express the human sodium iodide symporter-green fluorescent protein fusion reporter (hNIS-GFP; MDA-MB-231.hNIS-GFP) as a model for controlled cellular radionuclide uptake. Uptake, efflux, and subcellular location of the NIS radiotracer [99mTc]TcO4- were characterised to calculate the nuclear-absorbed dose using Medical Internal Radiation Dose formalism. Radiotoxicity was determined using clonogenic and γ-H2AX assays. The daughter radionuclide technetium-99 or external beam irradiation therapy (EBRT) served as controls. [99mTc]TcO4- in vivo biodistribution in MDA-MB-231.hNIS-GFP tumour-bearing mice was determined by imaging and complemented by ex vivo tissue radioactivity analysis. [99mTc]TcO4- resulted in substantial DNA damage and reduction in the survival fraction (SF) following 24 h incubation in hNIS-expressing cells only. We found that 24,430 decays/cell (30 mBq/cell) were required to achieve SF0.37 (95%-confidence interval = [SF0.31; SF0.43]). Different approaches for determining the subcellular localisation of [99mTc]TcO4- led to SF0.37 nuclear-absorbed doses ranging from 0.33 to 11.7 Gy. In comparison, EBRT of MDA-MB-231.hNIS-GFP cells resulted in an SF0.37 of 2.59 Gy. In vivo retention of [99mTc]TcO4- after 24 h remained high at 28.0% ± 4.5% of the administered activity/gram tissue in MDA-MB-231.hNIS-GFP tumours. [99mTc]TcO4- caused DNA damage and reduced clonogenicity in this model, but only when the radioisotope was taken up into the cells. This data guides the safe use of technetium-99m during imaging and potential future therapeutic applications.

In Vitro and Preclinical Systematic Dose-Effect Studies of Auger Electron- and ß Particle-Emitting Radionuclides and External Beam Radiation for Cancer Treatment.

PURPOSE: Despite a rise in clinical use of radiopharmaceutical therapies, the biological effects of radionuclides and their relationship with absorbed radiation dose are poorly understood. Here, we set out to define this relationship for Auger electron emitters [99mTc]TcO4- and [123I]I- and ß--particle emitter [188Re]ReO4-. Studies were carried out using genetically modified cells that permitted direct radionuclide comparisons. METHODS AND MATERIALS: Triple-negative MDA-MB-231 breast cancer cells expressing the human sodium iodide symporter (hNIS) and green fluorescent protein (GFP; MDA-MB-231.hNIS-GFP) were used. In vitro radiotoxicity of [99mTc]TcO4-, [123I]I-, and [188Re]ReO4- was determined using clonogenic assays. Radionuclide uptake, efflux, and subcellular location were used to calculate nuclear absorbed doses using the Medical Internal Radiation Dose (MIRD) formalism. In vivo studies were performed using female NSG mice bearing orthotopic MDA-MB-231.hNIS-GFP tumors and compared with X-ray-treated (12.6-15 Gy) and untreated cohorts. Absorbed dose per unit activity in tumors and sodium iodide symporter-expressing organs was extrapolated to reference human adult models using OLINDA/EXM. RESULTS: [99mTc]TcO4- and [123I]I- reduced the survival fraction only in hNIS-expressing cells, whereas [188Re]ReO4- reduced survival fraction in hNIS-expressing and parental cells. [123I]I- required 2.4- and 1.5-fold lower decays/cell to achieve 37% survival compared with [99mTc]TcO4- and [188Re]ReO4-, respectively, after 72 hours of incubation. Additionally, [99mTc]TcO4-, [123I]I-, and [188Re]ReO4- had superior cell killing effectiveness in vitro compared with X-rays. In vivo, X-ray led to a greater median survival compared with [188Re]ReO4- and [123I]I- (54 days vs 45 and 43 days, respectively). Unlike the X-ray cohort, no metastases were visualized in the radionuclide-treated cohorts. Extrapolated human absorbed doses of [188Re]ReO4- to a 1 g tumor were 13.8- and 11.2-fold greater than for [123I]I- in female and male models, respectively. CONCLUSIONS: This work reports reference dose-effect data using cell and tumor models for [99mTc]TcO4-, [123I]I-, and [188Re]ReO4- for the first time. We further demonstrate the tumor-controlling effects of [123I]I- and [188Re]ReO4- in comparison with external beam radiation therapy.

hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex.

hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the function of MRN. Strikingly, silencing of hSSB1 prevents foci formation as well as recruitment of MRN to sites of DSBs and leads to a subsequent defect in resection of DSBs as evident by defective RPA and ssDNA generation. Our data suggests that hSSB1 functions upstream of MRN to promote its recruitment at DSBs and is required for efficient resection of DSBs. These findings, together with previous work establish essential roles of hSSB1 in controlling ATM activation and activity, and subsequent DSB resection and homologous recombination (HR).

Enhanced effect of X-rays in the presence of a static magnetic field within a 3D pancreatic cancer model.

OBJECTIVE: To evaluate the impact of static magnetic field (SMF) presence on the radiation response of pancreatic cancer cells in polyurethane-based highly macro-porous scaffolds in hypoxic (1% O2) and normoxic (21% O2) conditions, towards understanding MR-guided radiotherapy, shedding light on the potential interaction phenomenon between SMF and radiation in a three-dimensional (3D) microenvironment. METHODS: Pancreatic cancer cells (PANC-1, ASPC-1) were seeded into fibronectin-coated highly porous polyethene scaffolds for biomimicry and cultured for 4 weeks in in vitro normoxia (21% O2) followed by a 2-day exposure to either in vitro hypoxia (1% O2) or maintenance in in vitro normoxia (21% O2). The samples were then irradiated with 6 MV photons in the presence or absence of a 1.5 T field. Thereafter, in situ post-radiation monitoring (1 and 7 days post-irradiation treatment) took place via quantification of (i) live dead and (ii) apoptotic profiles. RESULTS: We report: (i) pancreatic ductal adenocarcinoma hypoxia-associated radioprotection, in line with our previous findings, (ii) an enhanced effect of radiation in the presence of SMFin in vitro hypoxia (1% O2) for both short- (1 day) and long-term (7 days) post -radiation analysis and (iii) an enhanced effect of radiation in the presence of SMF in in vitro normoxia (21% O2) for long-term (7 days) post-radiation analysis within a 3D pancreatic cancer model. CONCLUSION: With limited understanding of the potential interaction phenomenon between SMF and radiation, this 3D system allows combination evaluation for a cancer in which the role of radiotherapy is still evolving. ADVANCES IN KNOWLEDGE: This study examined the use of a 3D model to investigate MR-guided radiotherapy in a hypoxic microenvironment, indicating that this could be a useful platform to further understanding of SMF influence on radiation.

Characterization of Inorganic Scintillator Detectors for Dosimetry in Image-Guided Small Animal Radiotherapy Platforms.

The purpose of the study was to characterize a detection system based on inorganic scintillators and determine its suitability for dosimetry in preclinical radiation research. Dose rate, linearity, and repeatability of the response (among others) were assessed for medium-energy X-ray beam qualities. The response's variation with temperature and beam angle incidence was also evaluated. Absorbed dose quality-dependent calibration coefficients, based on a cross-calibration against air kerma secondary standard ionization chambers, were determined. Relative output factors (ROF) for small, collimated fields (≤10 mm × 10 mm) were measured and compared with Gafchromic film and to a CMOS imaging sensor. Independently of the beam quality, the scintillator signal repeatability was adequate and linear with dose. Compared with EBT3 films and CMOS, ROF was within 5% (except for smaller circular fields). We demonstrated that when the detector is cross-calibrated in the user's beam, it is a useful tool for dosimetry in medium-energy X-rays with small fields delivered by Image-Guided Small Animal Radiotherapy Platforms. It supports the development of procedures for independent "live" dose verification of complex preclinical radiotherapy plans with the possibility to insert the detectors in phantoms.

Development and optimisation of a preclinical cone beam computed tomography-based radiomics workflow for radiation oncology research.

Background and purpose: Radiomics features derived from medical images have the potential to act as imaging biomarkers to improve diagnosis and predict treatment response in oncology. However, the complex relationships between radiomics features and the biological characteristics of tumours are yet to be fully determined. In this study, we developed a preclinical cone beam computed tomography (CBCT) radiomics workflow with the aim to use in vivo models to further develop radiomics signatures. Materials and methods: CBCT scans of a mouse phantom were acquired using onboard imaging from a small animal radiotherapy research platform (SARRP, Xstrahl). The repeatability and reproducibility of radiomics outputs were compared across different imaging protocols, segmentation sizes, pre-processing parameters and materials. Robust features were identified and used to compare scans of two xenograft mouse tumour models (A549 and H460). Results: Changes to the radiomics workflow significantly impact feature robustness. Preclinical CBCT radiomics analysis is feasible with 119 stable features identified from scans imaged at 60 kV, 25 bin width and 0.26 mm slice thickness. Large variation in segmentation volumes reduced the number of reliable radiomics features for analysis. Standardization in imaging and analysis parameters is essential in preclinical radiomics analysis to improve accuracy of outputs, leading to more consistent and reproducible findings. Conclusions: We present the first optimised workflow for preclinical CBCT radiomics to identify imaging biomarkers. Preclinical radiomics has the potential to maximise the quantity of data captured in in vivo experiments and could provide key information supporting the wider application of radiomics.

A systematic study of the contribution of counting statistics to the final lineal energy uncertainty in microdosimetry.

Objectives.Microdosimetry is proving to be a reliable and powerful tool to be applied in different fields such as radiobiology, radiation protection and hadron therapy. However, accepted standard protocols and codes of practice are still missing. With this regard, a systematic and methodical uncertainty analysis is fundamental to build an accredited uncertainty budget of practical use. This work studied the contribution of counting statistics (i.e. number of events collected) to the final frequency-mean and dose-mean lineal energy uncertainties, aiming at providing guidelines for good experimental and simulation practice. The practical limitation of current technologies and the non-negligible probability of nuclear reactions require careful considerations and nonlinear approaches.Approach.Microdosimetric data were obtained by means of the particle tracking Monte Carlo code Geant4. The uncertainty analysis was carried out relying on a Monte Carlo based numerical analysis, as suggested by the BIPM's 'Guide to the expression of uncertainty in measurement'. Final uncertainties were systematically investigated for proton, helium and carbon ions at an increasing number of detected events, for a range of different clinical-relevant beam energies.Main results.Rare events generated by nuclear interactions in the detector sensitive volume were found to massively degrade microdosimetric uncertainties unless a very high statistics is collected. The study showed an increasing impact of such events for increasing beam energy and lighter ions. For instance, in the entrance region of a 250 MeV proton beam, about 5 ∗ 107events need to be collected to obtain a dose-mean lineal energy uncertainty below 10%.Significance.The results of this study help define the necessary conditions to achieve appropriate statistics in computational microdosimetry, pointing out the importance of properly taking into account nuclear interaction events. Their impact on microdosimetric quantities and on their uncertainty is significant and cannot be overlooked, particularly when characterising clinical beams and radiobiological response. This work prepared the ground for deeper investigations involving dedicated experiments and for the development of a method to properly evaluate the counting statistics uncertainty contribution in the uncertainty budget, whose accuracy is fundamental for the clinical transition of microdosimetry.

3d tissue models as tools for radiotherapy screening for pancreatic cancer.

The efficiency of radiotherapy treatment regimes varies from tumour to tumour and from patient to patient but it is generally highly influenced by the tumour microenvironment (TME). The TME can be described as a heterogeneous composition of biological, biophysical, biomechanical and biochemical milieus that influence the tumour survival and its' response to treatment. Preclinical research faces challenges in the replication of these in vivo milieus for predictable treatment response studies. 2D cell culture is a traditional, simplistic and cost-effective approach to culture cells in vitro, however, the nature of the system fails to recapitulate important features of the TME such as structure, cell-cell and cell-matrix interactions. At the same time, the traditional use of animals (Xenografts) in cancer research allows realistic in vivo architecture, however foreign physiology, limited heterogeneity and reduced tumour mutation rates impairs relevance to humans. Furthermore, animal research is very time consuming and costly. Tissue engineering is advancing as a promising biomimetic approach, producing 3D models that capture structural, biophysical, biochemical and biomechanical features, therefore, facilitating more realistic treatment response studies for further clinical application. However, currently, the application of 3D models for radiation response studies is an understudied area of research, especially for pancreatic ductal adenocarcinoma (PDAC), a cancer with a notoriously complex microenvironment. At the same time, specific novel and/or more enhanced radiotherapy tumour-targeting techniques such as MRI-guided radiotherapy and proton therapy are emerging to more effectively target pancreatic cancer cells. However, these emerging technologies may have different biological effectiveness as compared to established photon-based radiotherapy. For example, for MRI-guided radiotherapy, the novel use of static magnetic fields (SMF) during radiation delivery is understudied and not fully understood. Thus, reliable biomimetic platforms to test new radiation delivery strategies are required to more accurately predict in vivo responses. Here, we aim to collate current 3D models for radiation response studies of PDAC, identifying the state of the art and outlines knowledge gaps. Overall, this review paper highlights the need for further research on the use of 3D models for pre-clinical radiotherapy screening including (i) 3D (re)-modeling of the PDAC hypoxic TME to allow for late effects of ionising radiation (ii) the screening of novel radiotherapy approaches and their combinations as well as (iii) a universally accepted 3D-model image quantification method for evaluating TME components in situ that would facilitate accurate post-treatment(s) quantitative comparisons.

Novel Anticancer and Treatment Sensitizing Compounds against Pancreatic Cancer.

The isolation of chemical compounds from natural origins for medical application has played an important role in modern medicine with a range of novel treatments having emerged from various natural forms over the past decades. Natural compounds have been exploited for their antioxidant, antimicrobial and antitumor capabilities. Specifically, 60% of today's anticancer drugs originate from natural sources. Moreover, the combination of synthetic and natural treatments has shown applications for (i) reduced side effects, (ii) treatment sensitization and (iii) reduction in treatment resistance. This review aims to collate novel and natural compounds that are being explored for their preclinical anticancer, chemosensitizing and radiosensitizing effects on Pancreatic Ductal Adenocarcinoma (PDAC), which is a lethal disease with current treatments being inefficient and causing serve side effects. Two key points are highlighted by this work: (i) the availability of a range of natural compounds for potentially new therapeutic approaches for PDAC, (ii) potential synergetic impact of natural compounds with advanced chemo- and radio-therapeutic modalities for PDAC.

On the Evaluation of a Novel Hypoxic 3D Pancreatic Cancer Model as a Tool for Radiotherapy Treatment Screening.

Tissue engineering is evolving to mimic intricate ecosystems of tumour microenvironments (TME) to more readily map realistic in vivo niches of cancerous tissues. Such advanced cancer tissue models enable more accurate preclinical assessment of treatment strategies. Pancreatic cancer is a dangerous disease with high treatment resistance that is directly associated with a highly complex TME. More specifically, the pancreatic cancer TME includes (i) complex structure and complex extracellular matrix (ECM) protein composition; (ii) diverse cell populations (e.g., stellate cells), cancer associated fibroblasts, endothelial cells, which interact with the cancer cells and promote resistance to treatment and metastasis; (iii) accumulation of high amounts of (ECM), which leads to the creation of a fibrotic/desmoplastic reaction around the tumour; and (iv) heterogeneous environmental gradients such as hypoxia, which result from vessel collapse and stiffness increase in the fibrotic/desmoplastic area of the TME. These unique hallmarks are not effectively recapitulated in traditional preclinical research despite radiotherapeutic resistance being largely connected to them. Herein, we investigate, for the first time, the impact of in vitro hypoxia (5% O2) on the radiotherapy treatment response of pancreatic cancer cells (PANC-1) in a novel polymer (polyurethane) based highly macroporous scaffold that was surface modified with proteins (fibronectin) for ECM mimicry. More specifically, PANC-1 cells were seeded in fibronectin coated macroporous scaffolds and were cultured for four weeks in in vitro normoxia (21% O2), followed by a two day exposure to either in vitro hypoxia (5% O2) or maintenance in in vitro normoxia. Thereafter, in situ post-radiation monitoring (one day, three days, seven days post-irradiation) of the 3D cell cultures took place via quantification of (i) live/dead and apoptotic profiles and (ii) ECM (collagen-I) and HIF-1a secretion by the cancer cells. Our results showed increased post-radiation viability, reduced apoptosis, and increased collagen-I and HIF-1a secretion in in vitro hypoxia compared to normoxic cultures, revealing hypoxia-induced radioprotection. Overall, this study employed a low cost, animal free model enabling (i) the possibility of long-term in vitro hypoxic 3D cell culture for pancreatic cancer, and (ii) in vitro hypoxia associated PDAC radio-protection development. Our novel platform for radiation treatment screening can be used for long-term in vitro post-treatment observations as well as for fractionated radiotherapy treatment.

Evaluation of a micro ionization chamber for dosimetric measurements in image-guided preclinical irradiation platforms.

Image-guided small animal irradiation platforms deliver small radiation fields in the medium energy x-ray range. Commissioning of such platforms, followed by dosimetric verification of treatment planning, are mostly performed with radiochromic film. There is a need for independent measurement methods, traceable to primary standards, with the added advantage of immediacy in obtaining results. This investigation characterizes a small volume ionization chamber in medium energy x-rays for reference dosimetry in preclinical irradiation research platforms. The detector was exposed to a set of reference x-ray beams (0.5-4 mm Cu HVL). Leakage, reproducibility, linearity, response to detector's orientation, dose rate, and energy dependence were determined for a 3D PinPoint ionization chamber (PTW 31022). Polarity and ion recombination were also studied. Absorbed doses at 2 cm depth were compared, derived either by applying the experimentally determined cross-calibration coefficient at a typical small animal radiation platform 'user's' quality (0.84 mm Cu HVL) or by interpolation from air kerma calibration coefficients in a set of reference beam qualities. In the range of reference x-ray beams, correction for ion recombination was less than 0.1%. The largest polarity correction was 1.4% (for 4 mm Cu HVL). Calibration and correction factors were experimentally determined. Measurements of absorbed dose with the PTW 31022, in conditions different from reference were successfully compared to measurements with a secondary standard ionization chamber. The implementation of an End-to-End test for delivery of image-targeted small field plans resulted in differences smaller than 3% between measured and treatment planning calculated doses. The investigation of the properties and response of a PTW 31022 small volume ionization chamber in medium energy x-rays and small fields can contribute to improve measurement uncertainties evaluation for reference and relative dosimetry of small fields delivered by preclinical irradiators while maintaining the traceability chain to primary standards.

Impact of superparamagnetic iron oxide nanoparticles on in vitro and in vivo radiosensitisation of cancer cells.

PURPOSE: The recent implementation of MR-Linacs has highlighted theranostic opportunities of contrast agents in both imaging and radiotherapy. There is a lack of data exploring the potential of superparamagnetic iron oxide nanoparticles (SPIONs) as radiosensitisers. Through preclinical 225 kVp exposures, this study aimed to characterise the uptake and radiobiological effects of SPIONs in tumour cell models in vitro and to provide proof-of-principle application in a xenograft tumour model. METHODS: SPIONs were also characterised to determine their hydrodynamic radius using dynamic light scattering and uptake was measured using ICP-MS in 6 cancer cell lines; H460, MiaPaCa2, DU145, MCF7, U87 and HEPG2. The impact of SPIONs on radiobiological response was determined by measuring DNA damage using 53BP1 immunofluorescence and cell survival. Sensitisation Enhancement Ratios (SERs) were compared with the predicted Dose Enhancement Ratios (DEFs) based on physical absorption estimations. In vivo efficacy was demonstrated using a subcutaneous H460 xenograft tumour model in SCID mice by following intra-tumoural injection of SPIONs. RESULTS: The hydrodynamic radius was found to be between 110 and 130 nm, with evidence of being monodisperse in nature. SPIONs significantly increased DNA damage in all cell lines with the exception of U87 cells at a dose of 1 Gy, 1 h post-irradiation. Levels of DNA damage correlated with the cell survival, in which all cell lines except U87 cells showed an increased sensitivity (P < 0.05) in the linear quadratic curve fit for 1 h exposure to 23.5 µg/ml SPIONs. There was also a 30.1% increase in the number of DNA damage foci found for HEPG2 cells at 2 Gy. No strong correlation was found between SPION uptake and DNA damage at any dose, yet the biological consequences of SPIONs on radiosensitisation were found to be much greater, with SERs up to 1.28 ± 0.03, compared with predicted physical dose enhancement levels of 1.0001. In vivo, intra-tumoural injection of SPIONs combined with radiation showed significant tumour growth delay compared to animals treated with radiation or SPIONs alone (P < 0.05). CONCLUSIONS: SPIONs showed radiosensitising effects in 5 out of 6 cancer cell lines. No correlation was found between the cell-specific uptake of SPIONs into the cells and DNA damage levels. The in vivo study found a significant decrease in the tumour growth rate.

Radiation microbeams as spatial and temporal probes of subcellular and tissue response.

Understanding the effects of ionizing radiations are key to determining their optimal use in therapy and assessing risks from exposure. The development of microbeams where radiations can be delivered in a highly temporal and spatially constrained manner has been a major advance. Several different types of radiation microbeams have been developed using X-rays, charged particles and electrons. For charged particles, beams can be targeted with sub-micron accuracy into biological samples and the lowest possible dose of a single particle track can be delivered with high reproducibility. Microbeams have provided powerful tools for understanding the kinetics of DNA damage and formation under conditions of physiological relevance and have significant advantages over other approaches for producing localized DNA damage, such as variable wavelength laser beam approaches. Recent studies have extended their use to probing for radiosensitive sites outside the cell nucleus, and testing for mechanisms underpinning bystander responses where irradiated and non-irradiated cells communicate with each other. Ongoing developments include the ability to locally target regions of 3D tissue models and ultimately to target localized regions in vivo. With future advances in radiation delivery and imaging microbeams will continue to be applied in a range of biological studies.

Evaluating the Propagation of Uncertainties in Biologically Based Treatment Planning Parameters.

Biologically based treatment planning is a broad term used to cover any instance in radiotherapy treatment planning where some form of biological input has been used. This is wide ranging, and the simpler forms (e.g., fractionation modification/optimization) have been in use for many years. However, there is a reluctance to use more sophisticated methods that incorporate biological models either for plan evaluation purposes or for driving plan optimizations. This is due to limited data available regarding the uncertainties in these model parameters and what impact these have clinically. This work aims to address some of these issues and to explore the role that uncertainties in individual model parameters have on the overall tissue control probability (TCP)/normal tissue control probability (NTCP) calculated, those parameters that have the largest influence and situations where extra care must be taken. In order to achieve this, a software tool was developed, which can import individual clinical DVH's for analysis using a range of different TCP/NTCP models. On inputting individual model parameters, an uncertainty can be applied. Using a normally distributed random number generator, distributions of parameters can be generated, from which TCP/NTCP values can be calculated for each parameter set for the DVH in question. These represent the spread in TCP/NTCP parameters that would be observed for a simulated population of patients all being treated with that particular dose distribution. A selection of clinical DVHs was assessed using published parameters and their associated uncertainties. A range of studies was carried out to determine the impact of individual parameter uncertainties including reduction of uncertainties and assessment of what impact fractionation and dose have on these probabilities.

UK adaptive radiotherapy practices for head and neck cancer patients.

OBJECTIVE: To provide evidence on the extent and manner in which adaptive practices have been employed in the UK and identify the main barriers for the clinical implementation of adaptive radiotherapy (ART) in head and neck (HN) cancer cases. METHODS: In December 2019, a Supplementary Material 1, of 23 questions, was sent to all UK radiotherapy centres (67). This covered general information to current ART practices and perceived barriers to implementation. RESULTS: 31 centres responded (46%). 56% responding centres employed ART for between 10 and 20 patients/annum. 96% of respondents were using CBCT either alone or with other modalities for assessing "weight loss" and "shell gap," which were the main reasons for ART. Adaptation usually occurs at week three or four during the radiotherapy treatment. 25 responding centres used an online image-guided radiotherapy (IGRT) approach and 20 used an offline ad hoc ART approach, either with or without protocol level. Nearly 70% of respondents required 2 to 3 days to create an adaptive plan and 95% used 3-5 mm adaptive planning target volume margins. All centres performed pre-treatment QA. "Limited staff resources" and "lack of clinical relevance" were identified as the two main barriers for ART implementation. CONCLUSION: There is no consensus in adaptive practice for HN cancer patients across the UK. For those centres not employing ART, similar clinical implementation barriers were identified. ADVANCES IN KNOWLEDGE: An insight into contemporary UK practices of ART for HN cancer patients indicating national guidance for ART implementation for HN cancer patients may be required.

Corrigendum: Evaluating the Propagation of Uncertainties in Biologically Based Treatment Planning Parameters.

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Evaluation of a Novel Liquid Fiducial Marker, BioXmark®, for Small Animal Image-Guided Radiotherapy Applications.

BioXmark® (Nanovi A/S, Denmark) is a novel fiducial marker based on a liquid, iodine-based and non-metallic formulation. BioXmark® has been clinically validated and reverse translated to preclinical models to improve cone-beam CT (CBCT) target delineation in small animal image-guided radiotherapy (SAIGRT). However, in phantom image analysis and in vivo evaluation of radiobiological response after the injection of BioXmark® are yet to be reported. In phantom measurements were performed to compare CBCT imaging artefacts with solid fiducials and determine optimum imaging parameters for BioXmark®. In vivo stability of BioXmark® was assessed over a 5-month period, and the impact of BioXmark® on in vivo tumour response from single-fraction and fractionated X-ray exposures was investigated in a subcutaneous syngeneic tumour model. BioXmark® was stable, well tolerated and detectable on CBCT at volumes ≤10 µL. Our data showed imaging artefacts reduced by up to 84% and 89% compared to polymer and gold fiducial markers, respectively. BioXmark® was shown to have no significant impact on tumour growth in control animals, but changes were observed in irradiated animals injected with BioXmark® due to alterations in dose calculations induced by the sharp contrast enhancement. BioXmark® is superior to solid fiducials with reduced imaging artefacts on CBCT. With minimal impact on the tumour growth delay, BioXmark® can be implemented in SAIGRT to improve target delineation and reduce set-up errors.

Effects of Gadolinium MRI Contrast Agents on DNA Damage and Cell Survival when Used in Combination with Radiation.

Gadolinium is a commonly used contrast agent for magnetic resonance imaging (MRI). The goal of this work was to determine how MRI contrast agents affect radiosensitivity for tumour cells. Using a 225kVp X-ray cabinet source, immunofluorescence and clonogenic assays were performed on six cancer cell lines: lung (H460), pancreas (MiaPaCa2), prostate (DU145), breast (MCF7), brain (U87) and liver (HEPG2). Dotarem® contrast agent, at concentrations of 0.2, 2 and 20 mM, was used to determine its effect on DNA damage and cell survival. Measurements were performed using inductively coupled plasma mass spectrometry (ICP-MS) to determine the amount of gadolinium taken up by each cell line for each concentration. A statistically significant increase in DNA damage was seen for all cell lines at a dose of 1 Gy for concentrations of 2 and 20 mM, at 1 h postirradiation. At 24 h postirradiation, most of the DNA damage had been repaired, with approximately 90% repair for almost all doses of radiation and concentrations of Dotarem. Clonogenic results showed no statistically significant decrease in cell survival for any cell line or concentration. Uptake measurements showed cell line-specific variations in uptake, with MCF7 and HEPG2 cells having a high percentage uptake compared to other cell lines, with 151.4 ± 0.3 × 10-15 g and 194.8 ± 0.4 × 10-15 g per cell, respectively, at 2 mM Dotarem concentration. In this work, a variability in gadolinium uptake was observed between cell lines. A significant increase was seen in initial levels of DNA damage after 1 Gy irradiation for all six cancer cell lines; however, no significant decrease in cell survival was seen with the clonogenic assay. The observation of high levels of repair suggest that while initial levels of DNA damage are increased, this damage is almost entirely repaired within 24 h, and does not affect the ability of cells to survive and produce colonies.

Radiobiological Implications of Nanoparticles Following Radiation Treatment.

Materials with a high atomic number (Z) are shown to cause an increase in the level of cell kill by ionizing radiation when introduced into tumor cells. This study uses in vitro experiments to investigate the differences in radiosensitization between two cell lines (MCF-7 and U87) and three commercially available nanoparticles (gold, gadolinium, and iron oxide) irradiated by 6 MV X-rays. To assess cell survival, clonogenic assays are carried out for all variables considered, with a concentration of 0.5 mg mL-1 for each nanoparticle material used. This study demonstrates differences in cell survival between nanoparticles and cell line. U87 shows the greatest enhancement with gadolinium nanoparticles (2.02 ± 0.36), whereas MCF-7 cells have higher enhancement with gold nanoparticles (1.74 ± 0.08). Mass spectrometry, however, shows highest elemental uptake with iron oxide and U87 cells with 4.95 ± 0.82 pg of iron oxide per cell. A complex relationship between cellular elemental uptake is demonstrated, highlighting an inverse correlation with the enhancement, but a positive relation with DNA damage when comparing the same nanoparticle between the two cell lines.