Dosimetry and Biological Studies for Microbeam Radiation Therapy at the Canadian Light Source by Danielle Anderson A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Physics Department of Oncology University of Alberta © Danielle Anderson, 2015 Abstract Microbeam radiation therapy (MRT) is a pre-clinical type of radiation therapy that uses an array of high-dose microbeams to treat solid tumours. An intense, quasi- parallel synchrotron beam is collimated to create microbeams several 10s of µm wide, and separated by 100s of µm. Animal studies over the past two decades have demonstrated that the extreme spatial fractionation employed in MRT leads to an unusual normal tissue sparing, while being effective for tumour palliation, and in some cases, ablation. This work considers both physical and biological questions remaining in MRT, with a focus on preparation for MRT experimentation on the two BioMedical Imaging and Therapy (BMIT) beamlines at the Canadian Light Source (CLS). A variety of techniques and detectors were employed to investigate the geometric and relative dosimetric characteristics of the 05B1-1 and 05ID-2 beamlines as a basis for further dosimetry. The absolute air kerma rate on the 05B1-1 beamline was measured for several beam qualities (monoenergetic and filtered polyenergetic x-ray beams) using a cylindrical, variable-length free-air ionization chamber and Monte Carlo simulations were carried out to determine correction factors. Air kerma rates between 4.5 mGy/s and 5.2 Gy/s were measured. Additionally, reference dosimetry was performed using a cavity ionization chamber by applying a geometric correction based on the non-uniform beam profile and the ion chamber response function in the broad synchrotron x-ray beam. This allowed the determination of peak (at the most intense point in the beam) and mean air kerma rates for several beam qualities, with a range from 1.9 cGy/s to 1.9 Gy/s. ii The MRT dose distributions delivered by the 05ID-2 beamline were investigated theoretically using the Monte Carlo package PENELOPE. This work demonstrated that the 05ID-2 beamline has the necessary energy characteristics to provide the spatial fractionation and penetration required for MRT experimentation. The dose distributions in cubic head phantoms representing small, medium and large animals were also determined to understand the considerations required for moving from small (e.g., rodent) animal experimentation to larger (e.g., cat and dog) animals. The spatial fractionation of MRT dose distributions will necessitate unconventional methods for treatment plan optimization. To explore this requirement, four dose-volume metrics, the peak-to-valley dose ratio, the peak-to-mean-valley dose ratio, the mean dose and the percentage volume below a threshold dose, were explored with changing microbeam array geometry and phantom size. To investigate the DNA damage response in cell cultures to synchrotron- generated microbeams, the formation of γH2AX foci (a marker of DNA double-strand breaks), rates of foci clearance and apoptosis in cultured normal human fibroblasts and malignant glioma cells were examined on the 05B1-1 beamline. The two cell types demonstrated similar trends in γH2AX foci formation and clearance with dose and time after irradiation. Additionally, despite elevated levels of γH2AX foci at late times (up to 72 hours after irradiation), both cell types showed very low levels of apoptosis. The results also highlighted the importance of understanding the DNA damage response specific to cell type, and the consideration of non-apoptotic responses even at high doses. iii The research in this thesis establishes a foundation in experimental dosimetry, theoretical dosimetry, and cell culture studies for future MRT research on the BMIT beamlines at the Canadian Light Source. iv Preface The entirety of this thesis work represents original research motivated by the objectives of my Ph.D. project. This research was supervised by Drs. E.A. Siegbahn, B.G. Fallone, and B. Warkentin (primary supervisor). The research was made possible with the significant additional input and assistance of several collaborators. A summary of these contributions is given in the following. The characterization of the BioMedical Imaging and Therapy beamlines and cavity and free air ionization chamber dosimetry described in Chapters 3, 4 and 5 represents work that I performed, initiated and led. This work included a large amount of data collection and analysis. B. Warkentin assisted with the collection and interpretation of this data, and in project conceptualization; he also provided project guidance and oversight. E.A. Siegbahn and B.G. Fallone provided general guidance. Dr. M. McEwen, Dr. E. Mainegra-Hing, and Mr. H. Shen of the National Research Council of Canada assisted with various aspects of the free air ion chamber work, including initial chamber testing, aperture fabrication, and expert guidance on Monte Carlo simulation of device response. A slightly altered version of Chapter 6 was published as D.A. Anderson, E.A. Siegbahn, R. Serduc, B.G. Fallone, and B. Warkentin, “Evaluation of dose-volume metrics for microbeam radiation therapy dose distributions in head phantoms of various sizes using Monte Carlo simulations, “ Phys. Med. Biol. 57, 3223-48 (2012). I was responsible for the computer simulations, analysis of data, and manuscript composition. E.A. Siegbahn conceived of the initial study as well as the initial version of the simulation code and manuscript edits. R. Serduc and B.G. Fallone provided manuscript edits and general guidance. B. Warkentin was the supervising author, involved in concept and methodology development, manuscript edits, and project oversight. The material presented in Chapter 7 has been published in two articles: D.L. Anderson, R. Mirzayans, B. Andrais, E.A. Siegbahn, B.G. Fallone and B. Warkentin, ‘Spatial and temporal distribution of γH2AX fluorescence in human cell cultures following synchrotron-generated X-ray microbeams: lack of correlation between persistent γH2AX foci and apoptosis,” J. Synch. Rad. 21, 801-810 (2014), and D. v Anderson, B. Andrais, R. Mirzayans, E.A. Siegbahn, B.G. Fallone and B. Warkentin, “Comparison of two methods for measuring γ-H2AX nuclear fluorescence as a marker of DNA damage in cultured human cells: applications for microbeam radiation therapy,” J. Inst. 8, C06008 (2013). I led and was responsible for all aspects of the project, including: project conceptualization; handling (e.g. transport of equipment between the Canadian Light Source (Saskatoon, SK) and the Cross Cancer Institute (Edmonton, AB), growing, irradiating, immunostaining and imaging our cell culture samples; developing original software for image analysis; and manuscript composition. B. Andrais assisted in planning the logistics of the experiments, initiating cell culture and preparing materials for transport, and assisted in immunostaining. R. Mirzayans was involved in concept formation, experiment planning, imaging the samples, analyzing and interpreting the data, and manuscript composition. E.A. Siegbahn and B.G. Fallone were involved with manuscript edits and general guidance. B. Warkentin was responsible for project conceptualization and oversight, and assisted with methodology development, sample irradiation, and other guidance. The literature review and description of the Canadian Light Source in Chapters 1 and 2, as well as the concluding Chapter 8 were conceived and written independently, with editorial suggestions from my supervisors and supervising committee. vi Acknowledgements There are many people that have been invaluable in the completion of this research, and my general medical physics education. First, I am incredibly thankful for the guidance, supervision and constant encouragement provided by Dr. Brad Warkentin. Dr. Warkentin far exceeded his responsibilities as a supervisor in every way possible. He not only cared about my success within the graduate program, but also my overall happiness. I would also like to thank my two co-supervisors. Dr. Albert Siegbahn offered expertise in the field of microbeam radiation therapy (MRT), and also facilitated my introduction to the international MRT community, which greatly enriched my research experience. Dr. Siegbahn consistently provided helpful comments, and always did so in a supportive manner. Dr. Gino Fallone’s vast experience and expertise in medical physics were important for directing the aims of this unique project, as well as my general development as a researcher in medical physics, and providing funding. A large portion of my research involved investigating the response of cell cultures to ionizing radiation, and this was only possible through the direct involvement of Dr. Razmik Mirzayans. Dr. Mirzayans was always very generous with his time – we had many discussions on biological concepts, literature, planning experiments, and interpreting results. In addition to the science that he taught me, he was also a great role model in terms of demonstrating dedication and integrity. I need to thank Bonnie Andrais for patiently teaching me laboratory skills, helping plan experiments, preparing the considerable amount of materials required for the biological experiments at the Canadian Light Source, and being a friend. Dr. Tomasz Wysokinski and Dr. George Belev, as well as the rest of the BioMedical Imaging and Therapy scientists and staff, were very important for completing the experimental measurements.