MODELLING RADIOBIOLOGY EFFECTS OF X-RAYS IN SMALL LABORATORY ANIMALS TO DEVELOP GUIDELINES FOR PRECLINICAL COMPUTED TOMORGRAPHY IMAGING

Images acquired by X-rays provide doctors with anatomical information and assist in diagnoses or progression of health problems. In a similar manner, this imaging technique is used for clinical and preclinical research. When used by doctors or in clinical research the amount of X-ray (ionizing radiation) dose a person receives is regulated. This is not true in preclinical research where overexposure to ionizing radiation occurs since X-ray doses are unknown, unregulated and there are no guidelines. Overexposure potentially causes unnecessary suffering to animals and may impact research results, especially in longitudinal studies. The goal of this project is to set X-ray (CT) dose guidelines in order to minimize or eliminate any animal suffering and reduce the number of animals used by refining CT imaging experimental methods. It has been known for years that ionizing radiation causes cell/DNA damage. For instance, ionizing radiation is used as a cancer treatment to kill cells.
Worldwide Cancer Research and Cancer Research UK are advocating for continued preclinical cancer research but seek improved, more reliable results. A major part of preclinical cancer research is drug development, testing, tumour treatment and radiotherapy. This type of research generally includes CT, PET/CT or SPECT/CT. Reviewing 10 of the most recent preclinical cancer studies using rodents (>536,000, 5yrs) the average per study was n=60 rodents. This equates to >32,000,000 rodents. Locally, 250 rodents were used in PET/CT research studies over 1 year. Refining the CT method potentially would reduce the number of rodents used by 20% to 200 rodents. Using the 20% metric and similar research trend, the potential to save >6,000,000 rodents exits in cancer studies. Furthermore, drug research extends beyond cancer treatment with over 286,000 preclinical studies done in the last 5 years. With the same metrics and n=50, >14,000,000 rodents could be reduced to <12,000,000 in drug research. Along with the demand for increased research the demand for understanding the impact of X-rays is needed.
Understanding can be gained with well-established radiation simulation software used routinely in clinical research and cancer treatment planning. Simulation tools have 20 years of valid, reliable and consistent predictions with dose calculation algorithms accuracies better than 1%, providing details on organ, cell and DNA ionizing radiation damage. This project is designed to use these simulation techniques to evaluate the impact of preclinical ionizing radiation along with X-ray beam measurements for validation. Radiation simulations completely replace animals, determine radiation thresholds and set foundational preclinical CT dose regulations. Knowing the biological effects of preclinical X-ray doses provides answers, refines CT experimental methods, improves robustness and reliability of outcomes; reducing number of animals.
Additionally, last year >100,000 rodents received a CT, PET/CT or SPECT/CT in studies unrelated to cancer or drug research. This demonstrates CT is widely used, potentially 100,000 rodents may have suffered unnecessarily and possibly 20,000 rodents weren't needed. The preclinical research community recognizes the need for and is pursuing improvements in imaging methods and ionizing standards. Recently, several avenues for dissemination of research in this regard opened up. The European Society for Molecular Imaging (ESMI) and the Society of Nuclear Medicine and Molecular Imaging set up specific committees and conference forums. This funding body offers publication support and dissemination of results through blogs and the F1000 gateway open access. A key opportunity for dissemination, policy making and implementation is in the EMSI STANDARD committee, which this applicant is a lead member. Results from this project will have a substantial future impact on preclinical imaging in the UK, Europe and USA. (Searches:ISI Web of Science 8/2019).

Main objective: Establish preclinical X-ray dose guidelines/regulations for the refinement of CT experimental methods, minimize potential suffering caused by overexposure of ionizing and reduce of the number of animals used. Study design replaces rodents with DNA damage simulations, phantom X-ray dose measurements and absorbed dose calculation. Aim 1: Evaluate DNA damage caused by energy deposited in cells from ionising radiation using Monte Carlo simulation and radiobiological models. Simulations represent current preclinical X-ray tube voltages (kVp), exposure times (ms) and projections acquired. Simulations include rat and mouse models with the option of varying CT focal points, amperage (mA), filtering and binning as deemed necessary for greater understanding of damage. Cell and DNA damage will be quantitative events of segment damage for classified as no damage, single strand break, or double strand break. Aim 2: Measure absorbed ionising radiation doses at preclinical tube voltage ranges using newly developed 3D printed anthropomorphic rodent phantom. For comparative analysis and dose guideline validation, measure absorbed ionized radiation at preclinical tube voltage ranges (35 to 80kVp) will be measured. Previous studies assessing DNA damage by ionized radiation have shown single/double strand damage is done with high doses and low doses. Low doses can cause damage by scatter directly to the cell or a neighbouring cell. Therefore, measurements will include additional CT parameters which are "low-dose" or soft X-rays with the ion chamber and verified by the nano-Dots. Additionally, projections and exposure times will be set at constant to check for linearity. Measured dose will be compared to simulation parameter doses. Aim 3: In conjunction with aims 1&2, calculate expected absorbed doses to specific rodent organs. Simulations parameters will include both settings from aim 1 & 2 with the intentions as noted in the Summary, absorbed radiation are in mGy.

Worldwide rodent preclinical CT imaging is a backbone/staple for multiple experimental groups. At the University of Edinburgh several studies are ongoing involving pharmacological/radiotracer development led by Dr Tavares in which PET/CT is the imaging tool used. From start of research to regulatory approval, a new therapeutic drug typically takes 10 to 15 years before it reaches the pharmacist. However, the ability to improve the reliability of CT imaging data sets has the potential to immediately reduce that timescale, by a minimum of 2 years. According to the Tufts Center for the Study of Drug Development only approximately 1 in every 10,000 compounds gets through to a regulatory body. Refining imaging protocols not only speeds the R&D process up through the pipeline to humans but also reduces the number of animals and cost. Moreover, with over 286,000 therapeutic drug studies done in the last 5 years, the number of rodents used is staggering (ISI Web Science, 8/2019). If considering one of Dr Tavares's studies over the last year, using the refined CT protocol potentially there would have been a 20% reduction in the number of rodents used. Currently, Dr Tavares has four ongoing studies in which 36% will be impacted by the refinements, leading to a reduction of animals. This refinement increases confidence and success rate when identifying a drug compound for future development and streamlines the validation of its effectiveness. Thereby, reducing attrition rate for drug candidate selection and characterization prior to expensive lengthy human studies, reducing the R&D timescale, reducing rodents used.
As with pharmacological studies, cancer research utilizes CT. Cancer studies are carried out focusing on drug treatment, development and radiotherapy, which require assessing biological effect of drugs or treatment. In 2017 Jaffee et al. reported on the future cancer research priorities which included drug discovery and development. The report noted rodent "unbiased and rigorous biological studies, reproducibility and consistency " were needed [1]. Understanding a biological effect becomes difficult if the ionizing radiation the animal receives from a CT imaging is unknown. This ties in directly with this projects goal to understand the biological effects of ionizing radiation received during imaging. Providing a more rigorous, reproducible, robust and consistent results requires understanding and eliminating CT bias and the impact on animal welfare. Jaffee et al. also reported preclinical rodent cancer research models looking at "cell fate" dynamics, detection and technologies or strategies for single cell and cell-free DNA analysis was a priority [1]. Again, without knowledge of ionizing radiation cell damage by any CT imaging the impact on results are unknown. Understanding the dynamics of single strain/double strain DNA damage caused by ionizing radiation eliminates studies which are unnecessarily overdosing animals, potentially causing suffering, cumulative severity over longitudinal studies. Given over the last 5 years more than 536,000 cancer researcher studies were carried out. Taking the average number of rodents used from the 10 most recent studies the number of rodents imaged reaches over 32,000,000 (ISI Web Science, 8/2019). Using a refined CT method, the estimated potential reduction in the number of rodents used (base on metrics above, 20%) is to 25,600,000.
Ultimately, the lack of knowledge impacts animals and research results. This produces unreliable and non-translatable data to clinical trials; greater probability of failure, thus, starting the vicious cycle of additional preclinical rodent studies. Knowledge will provide improved research methods thereby reducing the number of rodents used and consequently eliminate or minimize any potential suffering experience by animals undergoing CT imaging across multiple preclinical sites and research fields.
[1]E. M. Jaffee et al. Lancet Oncol. 2017.